Background: Hepatitis B virus is a public health problem worldwide. WHO in 2015, two hundred fifty million people were living with chronic hepatitis B virus (HBV) infection worldwide, and that 900 000 had died from HBV infection, mostly as a result of cirrhosis or hepatocellular carcinoma. It is highly endemic in Asia and Sub-Saharan Africa. Hepatitis B virus is transmitted by vertical transmission, between family members within households by contact of non-intact skin or mucous membrane, unsafe sexual intercourse, transfusion of infected blood products, perinatal transmission, horizontal transmission, nosocomial infection and percutaneous inoculation, drug users. Perinatal and early childhood transmissions are the main routes of Hepatitis B virus infection in endemic areas like Ethiopia. The risk of infection transmission decreases with periodic perinatal screening, immunotherapy and hepatitis vaccine given both to the high risk mother and the newborn. Therefore, administration of hepatitis B immunoglobulin in combination with hepatitis B vaccines as post exposure prophylaxis is very important since vertical transmission rate is nearly 100% according to WHO. Even though there was an introduction of universal infants Hepatitis B virus immunization it remains a public health problem in Ethiopia and in our region specially our hospital and there is no study in our setting. Objective: To assess the proportion and factors associated with the of Hepatitis B virus infection among pregnant mothers who had ANC at TGCSH, Bahir Dar Ethiopia 2022. Methods: institutional based cross-sectional study was conducted from January-June, 2022 among pregnant women attending the antenatal clinic of Tibebe Gihone comprehensive specialized Hospital. After obtaining writhen informed consent, Data was collected using semi-structured questionnaire to assess proportion of hepatitis B virus infection and associated factors and some socio-demographic characteristics. Systematic random sampling technique was implemented to select 357 samples. Data entered to epidata version 3.1and exported to SPSS and analyzed using SPSS version 23. Binary logistic regression analysis was used to determine target variables at p value <0.25 to include in multivariate logistic regression. The result was considered statistically significant at p < 0.05 by multivariate logistic regression. Findings were presented using 95% CI of Crude Odds Ratios (COR) and Adjusted Odds Ratios (AOR).Model fitness was evaluated using the Hoshmer-Lemeshow goodness of fit test. The data were expressed by tables and pie chart. Result: The overall sero- prevalence of HBV infection was Twenty-three (6.4%) (95% CI: 4.1%–9.5%) which indicates intermediate endemicity. History of multiple sexual partner AOR = 11.3; 95% CI (3.5-26.5),history of STI AOR =6.7; 95%CI (1.8-24.1),,history of blood transfusion AOR =9.5; 95%CI (2.2-20.7), history of dental surgery [AOR =4.0; 95%CI (2.2-16.3),and women who live in urban AOR =5.1; 95%CI (1.1-22.4), had statistical significant association with HBsAg sero-positivity. Conclusion and recommendations: The proportion of hepatitis B infection is intermediate among pregnant women in the study area according to WHO classification Generally, the study can provide current prevalence estimate of Hepatitis B virus in our setting that could be vital for intervention to tackle the disease. Awareness creation on modes of transmission and early screening of all pregnant women attending antenatal care and who had medical/surgical history like blood transfusion and dental procedure must be strengthened to minimize and control infection. Keywords: hepatitis, hepatitis B, hepatitis B surface antigen, pregnant women.