Introduction: Hepatitis B virus (HBV) is a public health problem worldwide. It is highly endemic in Asia and Sub-Saharan Africa. Even though there was an introduction of universal infants HBV immunization in 2007, distribution of HBV remains a public health problem in Ethiopia. Objective: To determine the sero-prevalence of HBsAg among pregnant women and to identify Factors associated with the infection. Methods: A cross-sectional study was conducted from June 1-August 1, 2021 among pregnant women attending the antenatal clinic (ANC) of Wolkite Health Center. After obtaining written and informed consent, blood sample was collected from 270 pregnant women using consecutive non- duplicative sampling method. Serum was separated from whole blood and tested for using Bioline strip test. Data was collected using pre-structured questionnaire and analyzed using SPSS version 20. Multiple logistic regression analysis was used to determine the association between explanatory variables and the outcome variables. The result was considered statistically significant at p < 0.05. Result:In this study, 270 pregnant women were participated. The overall prevalence of sero-positive HBS Ag among pregnant women was 15(5.6%). The study participants having history of abortion [AOR=0.024; 95%CI (.003-.165), p=0.000] and multiple sexual practice [AOR=0.013, 95%Cl (.001-.154), p=0.001] significantly predictor of Hepatitis B virus infection. Conclusion: The seroprevalence of hepatitis B virus infection was increased among study participants. From assessed associated factor history of abortion and multiple sexual practice were significantly associated with sero-prevalence of HBV infection. Do to these reason routine screening of women for HBV infection during pregnancy and ANC follow up.