Background: Sensitive diagnostics are needed to improve management and surveillance of opisthorchiasis and opisthorchiasis-associated cholangiocarcinoma (CCA), given the major public health problem of this condition in East Asia. We aimed to screen the Opisthorchis viverrini surface and secreted recombinant proteomes to identity antibody biomarkers of liver fluke infection and fluke-associated bile duct cancer, validate their diagnostic performance with sera donated by study participants in endemic populations in Thailand and nearby regions, and evaluate their utility as point-of-care immunochromatographic tests (PoC-ICTs) for this infection and associated malignancy. Method: We carried out a biomarker identification study, where we constructed a proteome array containing 250 validated and predicted proteins from the O. viverrini secretome and screened the array with sera from endemic populations in Thailand, Laos and China. Arrayed antigens that were IgG-reactive for diagnosing infection as well as infection-associated CCA were expressed in Escherichia coli and purified recombinant proteins were incorporated into PoC-ICTs to assess their predictive values for diagnosing liver fluke infection and infected-associated CCA from sera. Findings: A total of 825 proteins were identified by mass spectrometry from the excretory/secretory (ES) products and distinct extracellular vesicle populations, and 245 proteins were selected for printing based on their abundance and presence in multiple samples. Arrayed antigens that were the target of diagnostically informative IgG responses were validated by ICT. Two of the most promising antigens, P1 cysteine protease and P9 isocitrate dehydrogenase were each incorporated into a field-deployable PoC-ICT to further validate their diagnostic performance. Significant correlations were observed between P1-IgG reactivity and fecal egg counts (r = 0.432, p < 0.001) and between P9-IgG4 reactivity and fecal egg counts (r = 0.450, p < 0.001). The P9-IgG4 ICT was superior amongst the tests that involved a single recombinant protein (or crude parasite extract), yielding a sensitivity of 76.3% and specificity of 87.5%. For diagnosing fluke-induced CCA, both P1-IgG and P9-IgG4 out-performed ES-IgG ICTs for their respective antibody subclasses, including in combination form; 75% of patients were positive for at least IgG or IgG4 against ES products whereas 92% of patients were positive for at least one of the P1-IgG (AUC 0.84) or P9-IgG4 ICTs. Indeed, P9-IgG4 ICT alone accurately identified 86% of the CCA cases. Interpretation: We identified two biomarkers of O. viverrini infection and infection-associated CCA that could form the basis of novel antibody serodiagnostic tests. Future studies will address the performance of pilot PoC-ICTs as field-deployable using finger prick blood from larger cohorts. This study represents a truly bench-to-field approach to advance the development of serodiagnostics for human liver fluke infection and associated cancer. Funding: This study was supported by grants from the National Research Council of Thailand (NRCT): High-Potential Research Team Grant Program (Contract no. N42A670561 to WM, PMI, LS, RR, PB), the Office of the Ministry of Higher Education, Science, Research, and Innovation under the Reinventing University 2024 Visiting Professor Program (WM), the Research Program from Research and Graduate studies, Khon Kaen University (KKU) (grant no. RP66-7-001 to WM), and by award CA164719 (TL, PMI, PLF, PJB, AL) from the National Cancer Institue, National Institues of Health (NIH), USA. AL is supported by an investigator grant from the National Health and Medical Research Council of Australia (2008450). Declaration of Interest: We declare no competing interests. Ethical Approval: Ethics approval for collection of samples from individuals from Thailand and Lao PDR was obtained from the Khon Kaen University Ethics Committee for Human Research (HE611507, HE631300, HE641114, HE641242 and HE664044). Clonorchiasis serum samples were approved by the Medical Ethics Committee of the National Institute of Infectious Diseases, Tokyo, Japan (Nos. 177 and 589). Collection and use of samples from individuals from Australia was approved by the James Cook University Human Research Ethics Committee under approval H8523. A de-identified panel of healthy control sera for protein microarray serology was collected at University of California Irvine General Clinical Research Center (GCRC), now Center for Clinical Research, under an approved protocol HS# 2007-5896. Excretory/secretory products of O. viverrini were obtained from flukes passaged through hamsters with approval from the Animal Ethics Committee of Khon Kaen University and according to the Ethics of Animal Experimentation of the National Research Council of Thailand (AEMDKKU 002/2018).