AIM: Prostate cancer (PCa) is a common malignant tumor in men. This study aimed to explore the predictive value of serum biomarkers combined with ultrasound parameters for postoperative Gleason grading in PCa. METHODS: This study included 65 PCa patients who underwent transurethral resection of the prostate in our hospital from January 2021 to December 2023. Based on postoperative Gleason grading, the study subjects were divided into a Mild Group (n = 34) and a Severe Group (n = 31). Levels of serological biomarkers such as total prostate-specific antigen (tPSA), free prostate-specific antigen (fPSA), and fPSA/tPSA (the ratio of fPSA to tPSA), along with ultrasound (US) parameters, including prostate-specific antigen density (PSAD), intraglandular prostate-specific antigen density (IGPSAD), extraglandular prostate-specific antigen density (EGPSAD), and US score were compared between the two groups to evaluate their predictive values for postoperative Gleason grading. RESULTS: The fPSA, tPSA, fPSA/tPSA ratio, PSAD, IGPSAD, EGPSAD, and US scores in the Severe Group were significantly different from those in the Mild Group (p < 0.05). The combined evaluation of fPSA, tPSA, fPSA/tPSA, PSAD, IGPSAD, EGPSAD, and US score for predicting postoperative Gleason grading in PCa patients revealed an area under the curve (AUC) of 0.947, indicating a better predictive performance. The AUC of multi-parameter union was significantly higher than that of individual parameters and serum indicators union (fPSA, tPSA and fPSA/tPSA) (p < 0.05). CONCLUSIONS: The combined use of multiple parameters (fPSA, tPSA, fPSA/tPSA, PSAD, IGPSAD, EGPSAD and US Score) shows better predictive performance than individual testing. The combined application has high predictive value in postoperative Gleason grading of PCa. These findings can effectively guide clinical decision-making and optimize diagnostic strategies for developing effective and targeted therapeutic interventions for PCa.