Background <p>Hepatitis B and C virus infection is a continuous public health problem in Ethiopia, with the prevalence of the disease significantly varying across administrative regions. Determining seroprevalence among people who are unaware of their infection status is essential for hepatitis diagnosis and treatment. We aimed to determine the seroprevalence of two hepatitis viruses among patients attending different service areas in a tertiary hospital in Ethiopia.</p> Methods <p>We conducted an institution-based retrospective study using serology laboratory data from January 2021 to December 2023 at Yekatit 12 Hospital Medical College in Addis Ababa. We retrieved hepatitis B virus and hepatitis C virus data from electronic databases. We ran a binary logistic regression analysis to assess the relationship between dependent and independent variables. Statistical significance was determined for variables having a P-value (<i>P</i> &lt; 0.05) and 95% confidence intervals.</p> Results <p>A total of 3332 and 9890 patients’ blood samples were screened for hepatitis B and C virus, respectively. The prevalence of hepatitis B was 5.4% (179/3332), and hepatitis C virus was 1.8% (175/9890). The overall prevalence of co-infection was 0.2% (7/3322). Hepatitis B virus prevalence fluctuated, while hepatitis C virus prevalence steadily increased from 2021 to 2023. The positivity rate of the hepatitis B virus is 5.4% (89/1643) and hepatitis C virus 1.86% (93/5414) among female patients. Age was significantly associated with hepatitis B and C virus infection. The two viruses have been found at a significantly higher prevalence rate among admitted and emergency department patients.</p> Conclusion <p>An intermediate seroprevalence of HBV and hepatitis C virus infection was detected among participants in our study area. Screening for hepatitis B and C can be beneficial in identifying people with viral hepatitis and offering a chance to engage them in specialized care.</p>