Background Hepatitis B virus (HBV) kills millions of people globally; it is worse in pregnant women. HBV and Human Immune Virus (HIV) co-infection is associated with increased liver diseases such as cirrhosis and hepatocellular carcinoma. This study aimed at identifying the determinants of HBV infection among HIV-positive pregnant women. Methods A multicentre unmatched case-control study was conducted among 109 cases (HBV/HIV co-infected) and 327 controls (HIV positive) pregnant women in seven hospitals of the Eastern Amhara region. Interview and chart review data collection techniques were employed by trained personnel. A binary logistic regression model was used to identify independent predictors of hepatitis B virus infection. Variables with a p-value of <0.05 and 95% confidence interval for odds ratio not containing 1 considered independent predictors of HBV infection. Results The findings of this study revealed that history of STI [AOR, 1.97, 95%CI, 1.09–3.56], hospital admission [AOR, 3.08, 95%CI, 1.69–5.61], traditional delivery care [AOR, 3.31, 95%CI, 1.72–6.37], family history of HBV [AOR, 3.33, 95%CI, 1.72–6.37], presence of opportunistic infections [AOR, 0.23, 95%CI, 0.12–0.58], viral load [AOR, 7.58, 95%CI, 3.18–8.01], CD4 count [AOR, 2.15, 95% CI, 1.01–4.59], anaemia [AOR, 3.07, 95% CI, 1.71–5.51] and unsafe sex [AOR, 1.98, 95%CI, 1.09–3.61] had a statistically significant association with HBV infection. Conclusions Several exposure variables had statistically significant association with HBV infection. High Viral Load appeared to be the largest predictor of HBV infection in HIV patients. Therefore, targeted interventions such as behavioral change intervention for unsafe sex and STI should be in place, and screening tests and treatment at the early stage of conception for both partners is necessary.