Hepatitis B is a well-recognized occupational risk for healthcare workers. This self-administered questionnaire study was designed to assess awareness and knowledge towards hepatitis B virus infection among 140 nurses at Serdang Hospital, Selangor, Malaysia from the period of 1st April to 30th September 2017. The response rate was 97.2% (n = 140/144). A total of 71.4% of participants showed adequate awareness of hepatitis B. Most participants had heard hepatitis B with the predominant sources receiving from friends, media and education. Majority of them did serology blood test of hepatitis B before. A total of 84.3% of participants were aware of vaccine available for hepatitis B, although 78.6% got vaccinated in the past. Antiviral treatment of hepatitis B was not well noticed by most of them. Education qualification determines the awareness of hepatitis B. Regarding the knowledge, 73.6% of participants showed poor knowledge of hepatitis B. Most participants understood that hepatitis B was caused by a virus infection and the organ most affected was the liver. Surprisingly, 77.9% of them failed to recognize that cancer could be caused by hepatitis B. Majority of participants were aware that transmission of hepatitis B could be mediated via sexual intercourse, as well as childbirth. Nevertheless, 14.3% of them believed that hepatitis B was able to spread by cough and sneeze. Older age, Chinese ethnicity, and having high educational qualification were factors leading to adequate knowledge of hepatitis B. Additional attention should be emphasized to strengthen knowledge towards hepatitis B among nurses and perhaps other healthcare workers in Malaysia.