Introduction: This survey was designed to investigate the knowledge awareness of obstetrics and gynaecology staff (Obs/Gyn staff) on the prevention of mother-to-child transmission (PMTCT) of hepatitis B virus (HBV). Methodology: Obs/Gyn staff from 21 of the 31 Chinese mainland provinces, who attended medical meetings or training classes from July to October 2011, were invited to complete a questionnaire regarding PMTCT of HBV. The questionnaire included the clinical implications of HBV serologic markers and PMTCT preventive measures for both pregnant women and infants. Results: A total of 828 questionnaires were distributed, 617 (74.5%) Obs/Gyn staff participated in the survey, and 559 (90.6%) questionnaires met the inclusion criteria. Overall, 90% of participants correctly determined the positive hepatitis B surface antigen (HBsAg) as infectious, but up to 27.5% mistakenly considered the presence of anti-HBe and/or anti-HBc with negative HBsAg as infectious. In total, 96.3% respondents knew that pregnant women should be screened for HBV infection, and 95.3% realized that infants of HBsAg-positive mothers should be injected with hepatitis B immunoglobulin and vaccine. On the other hand, with the available immunoprophylaxis, 13.8% participants mistakenly believed caesarean section may prevent HBV mother-to-child transmission, and only 13% correctly answered that newborns of HBsAg positive mothers may be breastfed. Conclusion: Obs/Gyn staff in China have mastered the strategies of HBV PMTCT, but there is obvious insufficiency in details of the application. Intensified efforts to train the Obs/Gyn staff are required to improve the current suboptimal medical service in HBV-exposed infants and to control mother-to-infant transmission of HBV.