We studied the association of the serum levels of the microRNA family members miR-320a/-b/-c with clinico-pathological data to assess their applicability as diagnostic biomarker in prostate cancer (PCa) patients. The levels of miR-320a/-b/-c in 3 groups were evaluated by qRT-PCR (145 patients with PCa, 31 patients with benign prostatic hyperplasia (BPH) and 19 healthy controls). The levels of the three family members of miR-320 were directly correlated within each group (P < 0.001), but they differed significantly among the three groups (P < 0.001). The serum levels of the miR-320 family members were significantly increased in older patients compared to younger patients (≤ 66 years vs. > 66 years, P ≤ 0.001). In addition, the levels of all three miR-320 family members were significantly different in patients with low tumor stage compared with those with high tumor stage (miR-320a: P = 0.034; miR-320b: P = 0.006; miR-320c: P = 0.007) and in patients with low serum PSA compared with those with high serum PSA (≤ 4 ng vs. > 4 ng; miR-320a: P = 0.003; miR-320b: P = 0.003; miR-320c: P = 0.006). The levels of these miRNAs were inversely correlated with serum PSA levels. Detection in the serum samples of PCa patients with or without PSA relapse revealed higher levels of miR-320a/-b/-c in the group without PSA relapse before/after radical prostatectomy than in that with PCa relapse.In summary, the differences among the PCa/BPH/healthy control groups with respect to miR-320a/-b/-c levels in conjunction with higher levels in patients without a PSA relapse than in those with a relapse suggest the diagnostic potential of these miRNA-320 family members in PCa patients.