Background: A significant outbreak of Murray Valley encephalitis virus (MVEV), an arthropod-borne flavivirus, was observed in Australia in 2023. This study provides a comprehensive characterisation of the 2023 Murray Valley encephalitis virus (MVEV) outbreak, the largest outbreak of this virus since 1974, focusing on utility of diagnostic platforms, testing algorithms and genomic characteristics. Methods: A nationwide case series of Murray Valley encephalitis (MVE) cases from 1 January to 31 July 2023 was collated across all Australian state-based arbovirus reference laboratories. Multimodal diagnostic frameworks incorporated individual flavivirus-specific serology and nucleic acid amplification testing. Findings: Our case series identified 27 MVE cases, affecting all age ranges (6 weeks to 83 years) with a male preponderance (3.5:1). Incidence varied widely by geographic region, and was highest in the Northern Territory (3.2 per 100,000 population). MVE diagnosis was achieved with appropriate specimen collection (median 6 days from symptom onset to diagnostic specimen collection, IQR 4 to 9 days), although the need for convalescent serum delayed case confirmation in a minority. MVEV-specific IgM was detectable in serum in 76% of patients by day 7 and MVEV IgG or total antibody in 100% by day 30. MVEV-specific IgM and MVEV RNA was detected in cerebrospinal fluid collected within 14 days of symptom onset in 36% and 30% patients, respectively. Phylogenetic analysis revealed two circulating MVEV genotypes in mosquitoes and humans, G1A and G2, with sole presence of G1A in Southeast Australia, most likely introduced from enzootic foci in northern Australia. Interpretation: This study provides a comprehensive overview of the 2023 MVE outbreak in Australia, emphasising the importance of a multimodal diagnostic approach for accurate and timely case confirmation. Further One Health surveillance for MVEV and other zoonotic flaviviruses is critical given potential expanded ecological niches in the context of episodic climatic events.