Objective To explore the relationship between microRNA-296-5p（miR-296-5p），S-phase kinase-associated protein 2（Skp2），ribosomal protein L6 （RPL6） and pathological characteristics of prostate cancer，as well as their impact on surgical prognosis. Methods A total of 120 patients with prostate cancer who were treated from January 2018 to January 2023 were enrolled，and all patients underwent laparoscopic therapy for prostate cancer. The mRNA expression levels of miR-296-5p，Skp2，and RPL6 were compared between the patients with different pathological features，and the association between each indicator and pathological features was analyzed. According to the presence or absence of recurrence at 1 year after surgery，the patients were divided into recurrence group with 21 patients and non-recurrence group with 97 patients. The two groups were compared in terms of clinical data and the mRNA expression levels of miR-296-5p，Skp2，and RPL6，and the influence of miR-296-5p，Skp2，and RPL6 on surgical prognosis and their predictive value were analyzed. Results There were statistically significant differences in the expression levels of miR-296-5p，Skp2，and RPL6 mRNA among patients with different pathological stages，Gleason scores，preoperative serum prostate specific antigen（PSA） levels，and lymph node metastasis（F=12.927，22.416，10.088，34.239，20.180，12.208；t=4.649，-5.770，-5.713，4.716，-5.647，-6.884；P=0.000 for all）. miR-296-5p was negatively correlated with Gleason score，pathological stage，preoperative serum PSA level，and lymph node metastasis（r=-0.578，-0.539，-0.517，-0.556；P<0.001 for all），while Skp2 and RPL6 mRNA were positively correlated with Gleason score，pathological stage，preoperative serum PSA level，and lymph node metastasis（r=0.531，0.507，0.476，0.493，0.494，0.473，0.420，0.505；P<0.001 for all）. Compared with the non-recurrence group，the recurrence group had significantly higher lymph node metastasis，pathological stage，rate of positive margin，and Gleason score and a significantly lower rate of postoperative adjuvant therapy（P<0.05）. Compared with the non-recurrence group，the recurrence group had a significantly higher expression level of miR-296-5p and significantly lower mRNA expression levels of Skp2 and RPL6（P<0.05）. Before correction，the logistic regression analysis showed that pathological stage，Gleason score，lymph node metastasis，positive surgical margin，postoperative adjuvant therapy，and the mRNA expression levels of miR-296-5p，Skp2，and RPL6 mRNA were influencing factors for postoperative biochemical recurrence in patients with prostate cancer（P<0.05），and after correction，the mRNA expression levels of miR-296-5p，Skp2，and RPL6 were still influencing factors for postoperative biochemical recurrence in patients with prostate cancer （P<0.05）. The ROC curve analysis showed that the combination of miR-296-5p，Skp2，and RPL6 had an area under the ROC curve of 0.902（95%CI=0.833～0.949） in predicting biochemical recurrence after prostate cancer surgery，with a significantly larger AUC than each indicator alone（P<0.05）. Conclusion The expression levels of miR-296-5p，Skp2，and RPL6 are closely associated with pathological features and postoperative recurrence in patients with prostate cancer，and the combination of the three indicators has a high reference value in predicting biochemical recurrence after prostate cancer surgery.