Objective  To explore the value of magnetic resonance imaging (MRI) combined with serum phosphoglycerate kinase 1 (PGK-1), DEAD-box RNA helicase 5 (DDX5) and prostate-specific antigen (PSA) in the diagnosis of prostate cancer.Methods  The observation group consisted of 120 patients with prostate cancer who received treatment at our hospital from October 2020 to December 2022, while the control group included 120 patients with prostate hyperplasia during the same period. All subjects underwent MRI examination, serum levels of PGK-1, DDX5 and PSA were measured by enzyme linked immunosorbent assay (ELISA). Serum PSA was detected by E601 electrochemiluminescence analyzer. The diagnostic value of serum PGK-1, DDX5 and PSA levels in prostate cancer was analyzed by receiver operating characteristic (ROC) curve. The diagnostic value of MRI combined with serum PGK-1, DDX5 and PSA detection for prostate cancer was analyzed using a four-grid table.Results  Compared with the control group, the serum levels of PGK-1, DDX5 and PSA in the observation group were obviously increased (all P<0.05). The levels of serum PGK-1 and DDX5 in patients with prostate cancer were correlated with TNM stage, differentiation, lymph node metastasis, and Gleason pathological grade (all P<0.05), the area under the ROC curve (AUC) of PGK-1, DDX5 and PSA in the diagnosis of prostate cancer was 0.867, 0.836 and 0.812, respectively. The diagnostic efficacy of combined serum PGK-1, DDX5 and PSA was higher than that of MRI, serum PGK-1, DDX5 and PSA alone.Conclusions  The serum levels of PGK-1, DDX5 and PSA in patients with prostate cancer increase, they are related to the clinicopathological characteristics of the patients. The combination of the two with MRI can improve the diagnostic value of prostate cancer.