Denmark is a country with low prevalence and incidence of blood borne viral infections. Among health care workers (HCWs) vaccination for hepatitis B is only offered to high-risk groups. The aims of this cross sectional survey were to determine the prevalence of hepatitis B, -C, and human immunodeficiency virus (HIV) among the staff at a Danish University hospital and to correlate this with risk factors for transmission. Additionally, we wanted to examine the current frequency of blood exposure, reporting habits and hepatitis B vaccination status in the staff. Of 1439 eligible hospital staffs included, 960 (67%) were HCWs. The overall human immunodeficiency virus (HIV)-, hepatitis C Virus (HCV)- and hepatitis B Virus (HBV)-prevalence was 0% (0/1439), 0.14% (2/1439) and 1.6% (23/1439), respectively. Twenty-three percent of HCWs were vaccinated against HBV. Age, blood transfusion and stay in endemic areas were associated independently to HBV infection as opposed to job-category, duration of employment, HBV vaccination status and blood exposure. Based on a 4-week recall period, the incidence of percutaneous blood exposure was 1.5/person-year. In conclusion the HIV and hepatitis prevalence was low despite frequent blood exposure and the principal risk factors were unrelated to work. Danish HCWs do not seem to be at increased risk of hepatitis B even though universal HBV vaccination has not been implemented.