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Background Hepatitis B and C viruses are the major public health concerns of the globe. The two hepatotropic viruses share common modes of transmission and their co-infection is common. Despite the provision of an effective prevention mechanism, the infections caused by these viruses remain a significant problem worldwide, particularly among developing countries like Ethiopia. Methods This institutional-based retrospective study was conducted between January 2014 December and December 2019 from documented laboratory logbooks of Adigrat general hospital serology laboratory, Tigrai, Ethiopia. data were collected and checked for completeness on a daily based, coded, entered, and cleaned using Epinfo version 7.1, exported and analyzed using SPSS version 23. Binary logistic regression analysis and Chi-square test (X2) assessed the association between dependent and independent variables. The corresponding variables with a P-value (P Results Out of 20,935 clinically suspected individuals, 20,622 were given specimens and tested for hepatitis B and C viruses with total completeness of 98.5%. The overall prevalence of hepatitis B and hepatitis C virus was found to be 3.57% (689/19,273) and 2.13% (30/1,405), respectively. The positivity rate of the hepatitis B virus was 8.0% (106/1317) and 3.24% (583/17,956) among males and females, respectively. Additionally, 2.49%( 12/481) of males and 1.94% (18/924) of females were positive for hepatitis C virus infection. The overall prevalence of co-infection for both hepatitis B and hepatitis C virus was 7.4% (4/54). Sex and age were significantly associated with hepatitis B and C virus infection. Conclusions The overall prevalence of hepatitis B and C is low intermediate according to the WHO criteria. Although there was a fluctuating trend of hepatitis B and C through the years 2014–2019, the result shows moreover declining trend. Both hepatitis B and C share similar routes of transmission and affect all age categories but males were more highly affected than females. Therefore, awareness creation of the community about the methods of transmission, education about prevention, and control of hepatitis B and C virus infection, and improving coverage of youth-friendly services in health facilities should be strengthened.