Hepatitis B infection is a global public health challenge. The World Health Organization estimates a high global burden of 40% for hepatitis B among Health Care Workers (HCWs) and low uptake of hepatitis B vaccination especially in developing countries. This study was conducted to assess the uptake of hepatitis B vaccination by doctors in a Tertiary Health Institution, Benin City, Edo State. A descriptive cross sectional study design was utilized for this study, data collected was coded, entered and analyzed using SPSS version 16.0 with statistical significance set at p<0.05. This study identified a 50.6% Hepatitis B vaccination uptake among doctors which was significant associated with knowledge of injection safety (?2=6.683; p=0.035). There is a need to sensitize doctors regularly for improved Hepatitis B vaccination uptake through appropriate institutional framework put in place to ensure its compliance and safeguard the scarce Human resources for Health.