Hepatitis B virus infection is one of the leading causes of liver disease in the world. This study was conducted to determine the prevalence of HBV infection and associated risk factors among pregnant women in Northern Ethiopia using a cross-sectional study design. A total of 328 pregnant women were included in this study. Clinical and socio-demographic data of the pregnant women were collected using a structured questionnaire by nurses or midwives during their ANC visit. For the detection of HBsAg, 5 mL of venous blood was collected from the pregnant women; serum was separated in the health facilities from the whole blood and was transported to Tigray Public Health Research Institute for analysis using. The data were analyzed using SPSS software version 20.0. (IBM). Association of variables with HBV infection was determined with multivariate analysis and P < 0.05 was considered statistically significant. The mean age of the study participants was 25.45 ± 5.067. The overall prevalence rate of HBV infection among the pregnant women was 5.5%. A statistical association of HBV infection with risk factors was seen on participants, who were making unprotected sexual practices with multiple partners (AoR = 6.4, 95%CI, 2-21, P = 0.03), on those who had HBV-infected person in their family (AoR = 8, 95%CI, 1-58, P = 0.02), and on those who had undergone surgical procedures (AoR = 6.8, 95%CI, 1-32, P = 0.022).