Abstract Objectives. 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 recombinant proteome to identity antibody biomarkers of liver fluke infection and fluke-associated CCA with sera from study participants in endemic populations and evaluate their utility as point-of-care immunochromatographic tests (PoC-ICTs). Methods. We constructed a proteome array containing 245 mass spectrometry-validated proteins from the O. viverrini secretome and screened the array with sera from endemic populations. Antigens that were IgG-reactive for diagnosing infection as well as infection-associated CCA were recombinantly expressed and incorporated into PoC-ICTs to assess their predictive values for diagnosing liver fluke infection and infection-associated CCA from sera. Results. Two of the most promising antigens from the proteome array screen, P1 and P9, were each incorporated into PoC-ICTs 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 PoC-ICT was superior amongst the single recombinant antigen tests, yielding a sensitivity of 76.3% and specificity of 87.5%. For diagnosing fluke-induced CCA, both P1-IgG and P9-IgG4 out-performed parasite crude extract-IgG ICTs, 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. Conclusion. We identified two biomarkers of O. viverrini infection and infection-associated CCA that could form the basis of novel antibody serodiagnostic tests. This study represents a truly bench-to-field approach to advance the development of serodiagnostics for human liver fluke infection and associated cancer.