<b><i>Background:</i></b> The aim of this study was to evaluate the clinical value of 16 G biopsy needle in transperineal template-guided prostate biopsy (TTPB), compared with 18 G biopsy needle. <b><i>Methods:</i></b> The patients who underwent TTPB from August 2020 to February 2021 were randomized into 2 groups using a random number table. The control group (<i>n</i> = 65) and the observation group (<i>n</i> = 58) performed biopsy with 18 G (Bard MC l820) and 16 G (Bard MC l616) biopsy needles, respectively. Positive rate of biopsy, Gleason score, complications, and pain score were statistically analyzed. <b><i>Results:</i></b> The age, prostate volume, PSA, and the number of cores were comparable between the 2 groups. The positive rate of biopsy in the observation group was 68.9% (40/58), meanwhile the control group was 46.2% (30/65). There was statistical difference between the 2 groups (<i>p</i> = 0.011). Gleason score of the observation group (8 [7–9]) was higher than that of the control group (8 [6–9]) (<i>p</i> = 0.038). There was no significant difference in pain score and complications including hematuria, hematospermia, perineal hematoma, infection, and urinary retention between the 2 groups (<i>p</i> > 0.05). <b><i>Conclusions:</i></b> 16 G biopsy needle significantly improved the positive rates and accurately evaluate the nature of lesions, meanwhile did not increase the incidence of complications compared with 18 G biopsy needle.