Background: Hepatitis B infection is a common disease all over the world. According to its endemicity, countries are divided into three groups (high, intermediate, low). India falls in the intermediate endemicity zone (prevalence of 2–8% with an average of 4%). The risk of contracting hepatitis B virus (HBV) is four times greater for healthcare workers, compared to that of the general adult population. So, the study was aimed to assess knowledge, practice, and attitude of nursing officers regarding hepatitis B infection and its prevention, its association with selected variables, and correlation between knowledge and practice. Materials and methods: A cross-sectional survey was carried out among 180 nursing officers working in an ophthalmic center of a tertiary care hospital using 38 items, self-administered questionnaire with three sections for knowledge, practice, and attitude regarding HBV infection and its prevention. The data were collected in the morning and evening shift as per the subject's availability after obtaining a written consent from subjects. The analysis was performed using the statistical analysis software. Results: Among 180 respondents, 8% had poor knowledge whereas 92% showed adequate knowledge about HBV infection and its prevention. Poor knowledge was apparent in items like diet required in hepatitis infection (29%); otherwise, in rest all the areas, adequate knowledge was observed. Regarding practice, 14% had poor practices whereas 86% showed good practices. With regard to attitude, majority of the subjects had positive attitude for the items like risk of getting HBV infection (80%), importance of HBV vaccination (97%), and using universal precaution (98%). Uncertainty and disagreement showing negative attitude was observed in items like revealing the HBsAg+ve status at work (73%), sharing utilities with an infected person (71%), feeling stressed to take care of an infected patient (38%), fear of social isolation (80%), and vaccination for an HBV-infected patient (60%).