Background This study aimed to determine the effect of in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and individual factors on the sex ratio of offspring at birth in China.Methods This retrospective cohort study analyzed 6,700 infants (singletons and twins) who were born between December 2016 and December 2019 after fresh embryo transfer. The primary outcome measured was the secondary sex ratio (SSR) among live births.Results A total of 4,892 infants were born after cleavage-stage embryo transfer, and 1,808 infants were born after blastocyst-stage embryo transfer. The SSR for IVF was significantly higher than that for ICSI (SSR = 113 :100 vs. 92:100, P < 0.05). Male infertility significantly decreased the proportion of male offspring compared with non-male infertility. Multivariate logistic regression after adjusting for confounders revealed that neither the fertilization procedure nor the infertility cause had a significant effect on the SSR. The SSR of blastocyst-stage embryo transfer was higher than that of cleavage-stage embryo transfer, but the difference was not significant.Conclusions The live birth sex ratio in babies conceived using assisted reproductive technology was influenced by ICSI and male infertility, which may decrease the percentage of male offspring. Therefore, clinics should be aware of the gender bias when carrying out IVF / ICSI assisted pregnancies.