Purpose <p>To develop and validate a novel ultrasound scoring system (USS) for assisting cognitive fusion-targeted biopsy (cTB).</p> Methods <p>We prospectively collected a study cohort consisting of 452 patients with biopsy-naïve, PSA ≤ 20 ng/ml and their 531 Prostate Imaging Reporting and Data System (PI-RADS) v2.1 ≥ 3 lesions. All MRI regions of interest were scored as USS 0, 1, 2, and 3 for the corresponding lesion or region on TRUS. The cumulative cancer detection rate of the biopsy cores was assessed according to USS. Subgroup analysis was conducted to assess the csPCa detection rate following the re-stratification of PI-RADS using USS. Receiver operating characteristics (ROC) analysis was performed for USS, PI-RADS and USS + PI-RADS. The area under the curve (AUC), sensitivity, and specificity were calculated at the cut-off selected by the Youden index.</p> Results <p>The overall cancer detection rates for USS scores of 0 to 3 were 0% (0/67), 66% (111/166), 83% (176/210), and 100% (59/59), respectively. For USS 2 and USS 3 lesions, the detection rates in targeting the 3rd core (79%, <i>P</i> = 0.774) and 2nd core (93%, <i>P</i> = 0.125) did not significantly increase with subsequent biopsy cores. In the subgroup analysis, the csPCa positive rate for USS 0 was zero across all PI-RADS categories. In contrast, USS 1, 2, and 3 enhanced the csPCa positive rate within each PI-RADS strata. In ROC analysis, the AUC (95% CI) for the combined USS + PI-RADS 0.85 (0.82–0.89) outperformed PI-RADS 0.77 (0.73–0.81) alone (<i>P</i> &lt; 0.001). USS + PI-RADS sensitivity (95% CI) was 80.7% (75.6–84.9) compared to PI-RADS 72.5% (67.6–77.0).</p> Conclusion <p>In cTB, USS has good performance in cancer risk re-stratification, with higher USS scores correlating with an increased likelihood of cancer and improved diagnostic accuracy.</p> Clinical Trial Registration <p>No. 2023-272-002, July 14, 2023.</p> Graphical abstract <p></p>