Abstract Study question Does luteinized unruptured follicle affect the clinical outcome of natural cycles for single high-quality frozen-thawed blastocyst transfer (FBT) cycles? Summary answer Luteinized unruptured follicle negatively affects the pregnancy outcomes of single high-quality FBT. What is known already Recent evidence revealed that natural ovulation cycle with a corpus luteum for frozen-thawed embryo transfer is superior to hormone replacing therapy cycle in preventing early miscarriage and preeclampsia. However, it remains controversial whether a luteinized unruptured follicle of the natural cycle affect the pregnancy outcomes of FBT. Study design, size, duration This was a retrospective cohort study comparing the pregnancy outcomes of singe high-quality FBT among 283 cases of LUF cycles and 1083 cases of ovulation cycles between January 2015 to December 2020 in a private fertility center. The study was approved by the hospital's Ethics Committee. Participants/materials, setting, methods A natural cycle for FBT was performed for all included patients, and was categorized into LUF or ovulation group based on the continuously monitoring of transvaginal ultrasound. Pregnancy outcomes were compared between the two groups. Logistic regression analysis was performed to adjust for important confounders. P < 0.05 was considered statistically significant. Main results and the role of chance Compared to ovulation group, the LUF group was associated with higher proportion of female indication of IVF treatment. There were no statistically significant differences regarding the parental age at oocyte retrieval, body mass index (BMI), cycle rank, infertility duration, proportion of nulliparity and fertilization method, endometrial thickness, and estrogen levels. P levers were higher in the ovulation group than those of LUF (P =0.028). Logistic regression indicated that after controlling for potential confounders, ovulation group was associated with higher incidence of ongoing pregnancy (aOR 1.460, 95% CI: 1.107-1.924) and live birth (aOR 1.455, 95% CI: 1.102-1.919). Ovulation group also had higher clinical pregnancy rate (aOR 1.255, 95% CI: 0.952-1.656) and lower early miscarriage rate (aOR 0.654, 95% CI: 0.394-1.087), but not reach statistical significance. Our results suggested that LUF negatively affected pregnancy outcomes of single high-quality FBT. Limitations, reasons for caution The primary limitation of this study was its retrospective nature, and it was difficult to distinguish some confounding factors. Wider implications of the findings Clinicians should counsel couples about the negative effect of LUF on the pregnancy outcome of FBT, particularly for those with few high-quality embryos. Trial registration number 2018YFC1003900/2018YFC1003904, SZSM201502035