Japanese encephalitis (JE), a neurotrophic disease, was first recorded in the state of West Bengal, India in 1973. Since then JE is being reported every year from different districts. With a view to identify the JE cases accurately, a study was undertaken to detect the Japanese encephalitis virus (JEV) as the etiologic agent from the acute encephalitis syndrome (AES) cases and to identify its distribution in different districts. We report the results of 513 blood or cerebrospinal fluid samples referred/collected from the hospitalized AES cases. The samples were initially subjected to Mac-ELISA test followed by reverse transcriptase (RT)-PCR for the detection of IgM antibodies and the JEV genome, specific to E gene, respectively. Out of 513 samples referred/collected, 139 (27.1%) samples were reactive to JE IgM antibody. The remaining 374 samples were screened to select those which had a history of illness with a duration of ≤3 days. Only 147 samples were selected and tested, out of which 36 (24.5%) isolates were achieved and those were RT-PCR positive against the control JEV strain. Detection of IgM antibody to JE and the RT-PCR result confirms the active circulation of JEV in different districts of West Bengal and needs to be monitored carefully.