Cytokines are important modulators secreted by many cell types that are involved in multiple biological functions, including human embryo development and implantation. Recently, several studies have confirmed that cytokines in most body fluids are predictive of pathophysiological processes. The purpose of this study was to explore whether the cytokines in the culture medium of single pre-implantation blastocysts could be used as non-invasive predictors of embryo implantation potential in frozen embryo transfer (FET) cycles. Seventy-eight culture medium samples were collected on day 5 from single blastocysts with different morphological scores between January 2023 and December 2023. All the blastocysts were cryopreserved through vitrification and transferred in subsequent FET cycles. The expression levels of cytokines were detected and measured using the Luminex's xMAP assay. The Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis was performed to analyze predictors. Cytokine candidates were subjected to multivariate logistic regression analysis to build a prediction model for blastocyst implantation potential. Among the 6 cytokines screened by LASSO regression analysis, there were marked differences in the expression levels of IL-18 and TRAIL between the groups with and without pregnancy. None of the clinical variables were identified as influencing the blastocyst implantation. The ROC curve analyses demonstrated that joint assessment of IL-18 and TRAIL levels has better predictive ability than each separately. The measurement of the specific cytokines in pre-implantation blastocyst culture medium may provide a new direction for research focusing on developing a precise, non-invasive method for predicting of embryo implantation potential with high sensitivity and specificity.