Objective To validate the value of MRI and ultrasound three-dimensional matrix positioning technique in transperineal targeted prostate biopsy so as to improve the positive rate of prostate biopsy. Methods A total of 15 patients received transperineal targeted prostate biopsy using MRI and ultrasound three-dimensional matrix positioning technique + traditional systemic prostate biopsy from Feb. 2015 to Jun. 2015 in Changhai Hospital, Second Military Medical University. The 15 patients met the following criteria:(1) at least one negative prostate biopsy; (2) had the indications for performing re-biopsy; and (3) MRI showed suspected sites of prostate cancer (the PI-RADS scores were 4-5). Before biopsy, all the MRI images were reconstructed and the three-dimensional coordinates(X, Y, Z axis) were confirmed. After general anesthesia, systemic prostate biopsy was performed under the guidance of transrectal ultrasound. Then the transrectal ultrasound-guided biopsy was carried out according to the location in previous MRI images, which were located by the three-dimensional coordinates (X, Y, Z axis). Results Fourteen (93.3%) of the 15 patients were finally confirmed with prostate cancer, with 13 (86.7%) being positive only in targeted biopsy, and 6 (40.0%) being positive only in systemic biopsy. There was only 1 (6.7%) whose targeted biopsy result was negative and systemic biopsy result was positive, and there were 8 cases (53.3%) with positive targeted biopsy but negative systemic biopsy. Only 1 case (6.7%) was negative in both targeted biopsy and systemic prostate biopsy. There was significant difference in positive rates between the targeted biopsy and systemic biopsy (P=0.002). Conclusion Transperineal targeted prostate biopsy with MRI and ultrasound three-dimensional matrix positioning technique can accurately locate and target puncture the suspected site of prostate cancer in MRI images, and it has a higher diagnostic efficiency especially for the sites which are relatively easy to be missed by systemic prostate biopsy, but its performance still needs further large sample studies.