Background: Japanese encephalitis distribution all over India is not uniform, and there is diversity about incidence, occurrence and prevention strategy in different state of the country. Health care professionals are graduated differently in different parts of the country about managing health problems depending on local health needs so knowledge and perception towards Japanese encephalitis may not be uniform among them all over the country. Objective: Present study was carried out to assess the knowledge and perception about JE among Health care professionals in a tertiary care centre. Methods: A cross-sectional study carried out involving 103 health professionals working in various departments above the rank of first year junior residents in the same hospital for six months. Study was conducted after taking consent from participants using preformed self-administered questionnaire. Results: There were participants of position from junior resident of 2__ampersandsignamp;3 year (64.1%) to faculty (35.9%) and many (57.3%) were from clinical departments. Overall knowledge (68%) of study participants was good and perception was positive (58.3%). There was no variation in knowledge and perception across gender, clinical and para-clinical department (chi-square P__ampersandsignlt;0.05). Regarding perception towards disease, there was no much difference in resident doctors and faculty as well duration of experience. Conclusion: Health care professionals have optimized JE knowledge, but there is a need to improve their perception of JE.