Japanese encephalitis (JE) stands as a primary contributor to viral encephalitis cases. Its clinical manifestations, however, often coincide with other neuro-infectious diseases. This necessitates a clear understanding of diagnostic approaches to distinctively identify JE. Blood and cerebrospinal fluid are paramount samples for its diagnosis. Proper transportation of these samples is vital to maintain their integrity and avoid potential spread of the pathogen. The clinical criteria for AES also encompass potential JE cases. A laboratory-confirmed diagnosis becomes essential to affirm JE. Techniques for JE’s lab diagnosis include virus isolation, serological tests, plaque reduction neutralization test (PRNT), reverse transcriptase polymerase chain reaction (RT-PCR), and multiplex PCR. Notably, the IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is frequently employed as, in most cases, JEV IgM begins to manifest shortly postinfection—appearing in CSF within 4 days and in serum between 4 and 9 days following onset of symptoms. Nevertheless, serological tests might yield inaccurate results due to cross-reactivity among flaviviruses. Thus, the evolution of a definitive JE-specific diagnostic tool is imperative. A precise, timely diagnosis of JEV would further fortify our efforts in combating this severe neurotropic virus.