Objective  To investigate the value of transrectal ultrasound (TRUS)/magnetic resonance imaging (MR) targeted puncture combined with free prostatespecific antigen (FPSA), D-dimer (D-D) and testosterone in the diagnosis of prostate cancer (PCa).Methods  From January 2020 to December 2022,62 patients with PCa diagnosed in our hospital were selected as the PCa group, and 62 patients with benign prostatic hyperplasia (BPH) diagnosed in our hospital during the same period were selected as the BPH group. TRUS/MR targeted puncture was performed in PCa group, and serum FPSA, D-D and testosterone were detected in both groups. The serum levels of FPSA, D-D and testosterone in two groups were observed, and the serum levels of FPSA, D-D and testosterone in different types of patients in PCa group were analyzed. Using postoperative pathology as the gold standard, the diagnostic efficacy of TRUS/MR targeted puncture and serum FPSA, D-D, testosterone alone and in combination with PCa was analyzed. The correlation between Gleason score and TRUS/MR targeted puncture, serum FPSA, D-D and testosterone in PCa patients was analyzed.Results  The serum FPSA and D-D levels in PCa group were higher than those in BPH group, and serum testosterone levels were lower than those in BPH group (all P<0.001). The serum levels of FPSA and D-D in low-risk PCa patients were lower than those in medium-risk and high-risk PCa patients, and the serum levels of testosterone were higher than those in medium-risk and high-risk PCa patients (all P<0.05). The serum levels of FPSA and D-D in medium-risk PCa patients were lower than those in high-risk PCa patients, and the serum levels of testosterone were higher than those in high-risk PCa patients (all P<0.001). The sensitivity, specificity, positive predictive value and negative predictive value of TRUS/MR targeted puncture combined with FPSA, D-D and testosterone in the diagnosis of PCa were higher than those in the single and combined diagnosis (all P<0.05). Pearson correlation analysis showed that Gleason score was not correlated with TRUS/MR targeted puncture and serum FPSA level (r=0.127, 0.151, all P>0.05), but positively correlated with D-D level and negatively correlated with serum testosterone level respectively(r=0.526, -0.461, all P<0.05). Multivariate Cox regression analysis showed that FPSA≥6.83 ng/mL, D-D≥0.71 mg/L and testosterone ≤0.20 μg/L were independent risk factors for PCa (all P<0.05).Conclusions  The levels of serum FPSA, D-D and testosterone in PCa patients are abnormal, and TRUS/MR targeted puncture combined with FPSA, D-D and testosterone has high diagnostic efficacy in PCa.