To evaluate the impact of magnetic resonance imaging/transrectal ultrasound fusion-targeted prostate biopsy on the diagnosis of clinically significant prostate cancer using real-time three-dimensional ultrasound-based organ-tracking technology. The present study was a retrospective review of 262 consecutive patients with prostate-specific antigen of 7.1ng/mL (interquartile range 4.0-19.8). All patients received pre-biopsy magnetic resonance imaging and had a suspicious lesion for clinically significant prostate cancer. All patients underwent a combination of systematic biopsy (6 cores) and three-dimensional ultrasound-based magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy (2 cores). The positive rate of any cancer, positive rate of clinically significant prostate cancer, Gleason score and maximum cancer core length were compared between systematic biopsy versus magnetic resonance imaging/transrectal ultrasound fusion-targeted prostate biopsy. Overall, the positive rate of any cancer per patient was 61% (160/262) in systematic biopsy versus 79% (207/262) in magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy (P<0.0001); and that of clinically significant prostate cancer per patient was 46% (120/262) in systematic biopsy versus 70% (181/262) in magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy (P<0.0001). The positive rate of any cancer per core was 21.7% (330/1523) in systematic biopsy versus 68.6% (406/592) in magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy (P<0.0001), and that of clinically significant prostate cancer per core was 12.7% (193/1423) in systematic biopsy versus 60.3% (357/592) in magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy (P<0.0001). Adding systematic biopsy leads to 13 more cancer cases (5%). The distribution of Gleason score (6/7/8/9/10) was 59/71/23/6/1 in systematic biopsy versus 48/105/36/15/2 in magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy (P=0.005). The maximum cancer core length was 5mm (0.5-16) in systematic biopsy versus 8mm (1-19mm) in magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy (P<0.0001). Three-dimensional ultrasound-based magnetic resonance imaging/transrectal ultrasound fusion-targeted biopsy seems to be associated with a higher detection rate of clinically significant prostate cancer, with fewer cores than systematic random biopsy. However, significant cancer can still be detected by the systematic technique only. A combination of systematic biopsy with the targeted biopsy technique would avoid the underdiagnosis of clinically significant prostate cancer.