HBV infection is notorious and is endemic in Sudan. Health care workers are at particular increased risk for the infection. Their knowledge and acceptance of vaccination against the virus is of at most importance for the control of the disease. Objective: To study the knowledge and vaccination status among health care workers in some central hospitals in Sudan. Methodology: Data from different categories of health workers were collected using structure questionnaire after taking informed consent. Data were analyzed using Chi-Square and T-Test. Results: Around 96.22% of surgeons knew their increased risk for infection, and 71.69% of them knew vaccine prevention. The overall screening for the virus was 32.2%. Only 26.19% of those who received the vaccination had completed the doses. None vaccination due to a none specified cause was the main reason among doctors and nurses. Knowledge about risk and vaccination was very low among cleaning staff and none of them had vaccination. Conclusion:Although knowledge about HBV risk in our health care workers was reasonable -apart from cleaning staff-, the vaccination among them was not satisfactory. Efforts have to be augmented and special care for cleaning staff is mandatory.