Background: Prostate cancer remains the most common solid organ malignancy among males in the United States. Commercial biomarkers and multiparametric MRI have been utilized to better triage men with elevated PSA and determine patients most likely to have clinically significant prostate cancer (csPCa) upon prostate biopsy. We studied the combination of MRI with PSA-based and novel extracellular vesicle (EV)-based biomarkers to determine the optimal pre-biopsy testing to reduce the number of unnecessary biopsies. Methods: Men presenting to our clinic with elevated PSA (≥ 2 ng/ml) were prospectively enrolled from October 2019 to March 2022. All men underwent multiparametric MRI and blood draws to determine prostate health index (PHI) scoring, EV density, and PSA density. MRI-fusion biopsy was performed with both systematic and targeted prostate sampling, with clinicians blinded to prior blood testing results. Blood concentrations of prostate EV (ProsEVs) were measured by nanoscale flow cytometry using antibodies against PSMA and STEAP1. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated, and receiver operator characteristic (ROC) curves were constructed separately for all patients, those without prior diagnosis of PCa, and biopsy-naïve patients. Bootstrapping analysis was performed to provide more accurate assessment of the predictive ability of all combinations. Results: Ultimately, 175 men enrolled and underwent MRI, blood testing, and prostate biopsy. Among the 175 patients included, 101 (58%) had never undergone biopsy, 38 (22%) had a previous negative biopsy, and 36 (21%) were on active surveillance (AS) for GS6 disease. AUC of mpMRI (PI-RADS≥3) to predict clinically significant PCa was 0.66 with a sensitivity, specificity, NPV and PPV of 95.2%, 37.6%, 76.0% and 79.26%. MRI combined with STEAP1-EV density had an AUC of 0.77 with a sensitivity, specificity, NPV and PPV of 90.1%, 46.7%, 64.4% and 80.9%. A multivariable model including MRI, STEAP1-EV density, PSA density and PHI density outperformed other combinations with an AUC, sensitivity, specificity, NPV and PPV of 0.90, 89.2%, 68.7%, 72.8% and 88.1%. Conclusions: Combination of mpMRI with ProsEV and PSA derivatives demonstrated better predictive ability for csPCa on biopsy compared to imaging or serum testing alone. Inclusion of prostate volume with biomarkers further increased predictive ability which could lead to a decrease in number of unnecessary biopsies for men with benign conditions and indolent cancer.