Background: In resource- limited setting, co-infection between HIV and hepatitis B virus (HBV) poses important public health considerations. This cross-sectional study was undertaken with the aim of determining HBV seroprevalence patterns in urban blood banks. Methods: A cross-sectional study was conducted at an urban blood bank setting. A total of 1610 blood donors were enrolled, and 283 consecutive plasma samples with unknown HBsAg status were selected for risks factors. HBV seroprevalence was based on the Chemiluminescence method (Cobas® e601, Roche). Potential risk factors associated with overt HBV infection were assessed by calculating the crude and adjusted odds ratio, 95% confidence intervalley (95% CI) and p values. Results: Of 1610 participants, overall rate seroprevalence of HBsAg was 5.5% (95% CI: 4.36%–6.58%) ranging from 0.06% (95% CI: 0-0.18) (HCV) to 0.12% (95% CI: 0-0.30) (Syphilis). Seroprevalence of infection increased in older age groups (20-39 years) but men had a statistically significant higher prevalence of overt HBV infection than women (P=0.0001). The multivariate model showed the following to be predictors of HBV infection: male gender (OR=2.5 (95% CI 1.14-5.58), P= 0.02), first-time donor status (OR = 11.06, (95% CI 5.34-22.9), P= 0.01) and residence outside of Libreville (OR = 2.52, 95% CI 1.09-5.83), P=0.03). Conclusion: HB and co-infection are n o t common in Gabon. Intermediate seroprevalence was associated with male gender, first-time donor status and residence outside of Libreville. HCV and HBV infection among the younger age groups are becoming an alarming issue. Prevention and control of HBV infection are needed to reduce HBV transmission.