Objective: To investigate the diagnostic efficacy of targeted biopsy combined with regional systematic biopsy in prostate cancer (PCa) in patients with prostate imaging reporting and data system v2.1 (PI-RADS v2.1) 4-5. Methods: From January 2023 to October 2023, patients who underwent prostate biopsy for the first time with total prostate specific antigen (tPSA) ≤ 20 ng/mL and had a multi-parametric magnetic resonance imaging (mpMRI) PI-RADS of 4-5 in Peking University First Hospital were prospectively collected. All the patients underwent transrectal ultrasound-guided cognitive fusion targeted biopsy (3 cores) followed by systematic biopsy (12 cores). Various hypothetical biopsy schemes were defined based on different biopsy sites. The detection effectiveness of targeted biopsy combined with regional systematic biopsy and other biopsy schemes for prostate cancer were compared using Cochran's Q and McNemar tests. Results: A total of 255 patients were enrolled, of whom 204 (80.0%) were detected with prostate adenocarcinoma and 187 (73.3%) were clinically significant with prostate cancer (csPCa). The detection rate of PCa with targeted biopsy was significantly lower than that of targeted biopsy combined with 12-core system biopsy (77.3% vs. 80.0%, P=0.016), and 71.4% (5/7) of the missed patients was csPCa. There was no significant difference in the detection rate between targeted biopsy combined with 4-core regional system biopsy and 12-core system biopsy (P>0.999), and 1 case of csPCa and clinically insignificant prostate cancer (cisPCa) were missed. There was no significant difference in the detection rate of PCa between targeted combined regional system biopsy and targeted combined lateral or traditional 6-core system biopsy and the number of cores were reduced. Missed diagnosis of targeted biopsy was correlated with the maximum diameter of the lesion (OR=0.086, 95%CI: 0.013-0.562, P=0.010). For the patients with PI-RADS 5, only 1 case of PCa was missed in 122 cases by targeted biopsy alone. For patients with PI-RADS 4, 6 PCa cases were missed among the 133 patients with targeted biopsy alone, and 1 case of csPCa and cisPCa were missed by targeted biopsy combined with regional system biopsy. The statistics of positive core counts for different biopsy schemes indicated that targeted combined regional systematic biopsy had a higher proportion of positive cores second only to targeted biopsy alone. Conclusion: Targeted biopsy combined with regional systematic biopsy has high diagnostic efficacy in patients with PI-RADS 4-5 and can be considered as one of the improved schemes for combined biopsy. Targeted biopsy alone is also a feasible option for patients for patients with a PI-RADS score of 5.