• Higher levels in serum EVs than in whole serum.• Higher levels in PCa patients vs. BPH patients (3.85-fold, p = 0.0007) and healthy controls (4.06-fold, p = 0.0005).• EV miRNA levels associated with PSA ≥ 10, GS ≥ 8, and T3/T4 stages.• Higher levels in LPCa vs. mPCa: AUC = 0.8694, sensitivity = 80%, specificity = 87.1%. Here, KLK3 is linked to posterior cortical atrophy.