To investigate the relationship between the level of serum tumor markers and the expression of programmed cell death-ligand-1 (PD-L1) in breast cancer patients, and to explore the diagnostic value of serum tumor markers for the positive expression of PD-L1. The basic information, preoperative serum tumor markers and postoperative pathological results of 139 patients with breast cancer who received treatment for the first time in People’s Liberation Army (PLA) General Hospital between 2019 and 2020 were collected. The relationship between preoperative tumor markers and postoperative pathological PD-L1 positive expression in breast cancer patients was analyzed, and binary and multivariate Logistic regression model was established to explore the predictive ability of various tumor markers for the positive expression of PD-L1. The serum level of neuron-specific enolase (NSE) was positively correlated with the expression of PD-L1. The level of NSE was statistically different between the two groups of PD-L1 (PD-L1 (0%) and PD-L1 (≥1%)) (p < 0.05). Serum NSE level was better than carbohydrate antigen15-3 (CA15-3) in predicting the positive expression of PD-L1. The sensitivity and specificity were 0.786 and 0.545 when the cutoff value is 11.28, respectively. The area under receiver operating characteristic (ROC) curve (AUC) = 0.711. Serum NSE is an independent risk factor for positive expression of PD-L1.