Background: Given the popularity of prostate specific antigen (PSA) testing in recent years, the number of patients undergoing diagnostic prostate biopsy has increased. The transrectal ultrasound-guided (TRUS) biopsy is considered as the gold standard for prostate cancer detection, although has a low sensitivity. Objectives: The current study aimed at enhancing prostate cancer diagnosis using MRI-TRUS fusion biopsy in patients with negative history of TRUS biopsy. Methods: In the current study, patients undergone TRUS prostate biopsy with benign results that were candidates for repeat biopsy were recruited. After making the preparations, patients underwent magnetic resonance imaging (MRI)-TRUS fusion biopsy. Gleason score, the number of involved cores, perineural invasion, perilymphovascular invasion, and the percentage of core involvement were recorded. Results: Of the 191 patients, 70 (36.6%) had positive biopsies. The frequency of non-detectable cancers by targeted biopsy based on the level of cancer risk showed that at the very high-risk level, five (29.4%) and at high-risk level, two (11.7%) subjects were not recognized. The mean Gleason score in targeted (7.47 ± 0.99) and random (7.13 ± 1.04) positive biopsies showed a significant difference between the two groups (P = 0.045). Targeted biopsies are better than random ones to detect high-risk (33.9% vs 29.2%, P = 0.013 respectively) and very high-risk cancers (45.3% vs. 41.5%, P = 0.05 respectively). Conclusions: The combination of both biopsy approaches is suggested to offer a reliable method with high rate of tumor detection.