Background: Vaccination is effective to prevent hepatitis B virus (HBV) infection. However, there is still a risk of infection after vaccination. In clinical work, we found that newborns were positive for HBV surface antigen (HBsAg) after vaccination.Objectives: To determine the effect of hepatitis B vaccination on the detection of HBsAg trend in newborns.Methods: We collected data at birth, history of vaccination for hepatitis B, quantitative HBsAg results, and other information about newborns born in our hospital from July 2017 to July 2020. Serum samples from healthy neonates were randomly selected to be supplemented with recombinant hepatitis B vaccine on a concentration gradient, and HBsAg was measured quantitatively.Results: Data from 1417 neonates were included in the study; 306 (21.6%) were HBsAg positive within 8 d after vaccination, with levels ranging from 0.104 IU/mL to 0.339 IU/mL. The proportion of neonates with HBsAg-positive serum was significantly correlated with the level of hepatitis B surface antibodies (anti-HBs) in the serum of their mothers (P < 0.01). Experiments in vitro showed that the proportion of neonates with HBsAg-positive serum was correlated with the dose of the hepatitis B vaccine, and when the concentration of the hepatitis B vaccine reached 5 ng/mL and 10 ng/mL, the serum HBsAg levels showed a significant negative correlation with the original concentration of serum anti-HBs.Conclusions: Hepatitis B vaccination can affect the level of HBsAg detected in neonatal serum, and the effect could be mitigated by delaying the measurement. Moreover, maternal anti-HBs offset the effects of neonatal vaccination on HBsAg serum levels.