Trophectoderm biopsy is the most widely used biopsy method at present, its effectiveness and safety have always been a concern. Notably, various factors associated with the assisted reproductive technology (ART) process may be related to adverse maternal and neonatal outcomes. After taking potential factors into account, this study was designed to investigate whether trophectoderm biopsy increases the risk of adverse maternal and neonatal outcomes. This respective cohort study enrolled 3373 ICSI (Intracytoplasmic sperm injection) single frozen-thawed blastocyst transfer cycles with and without trophectoderm biopsy in the Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University in China from January 2015 to June 2020. Statistical methods including univariate logistic regression analysis, multivariate logistic regression analysis and stratified analysis were performed to analyze pregnancy and neonatal outcomes. We found that PGT (Preimplantation genetic testing) with trophectoderm biopsy does not increase the risk of adverse maternal and neonatal outcomes in ICSI single frozen-thawed blastocyst transfer cycles, and PGT can reduce the rate of birth defects. In addition, whether trophectoderm biopsy is performed or not, endometrial preparation by natural cycle and transplantation of Day 5 blastocysts and high-quality blastocysts may be conducive to better pregnancy outcomes, including higher rates of clinical pregnancy and live birth, as well as lower abortion rate.