To compare the expression of two promising circulating micro-ribonucleic acids (miRNAs 21 and 221) in patients with prostate cancer to subjects without cancer and to evaluate their potential role as specific noninvasive molecular biomarkers for prostate cancer diagnosis, circulating miRNAs 21 and 221 expression profiles were analyzed in 20 men aged 50-75 years, presenting with lower urinary tract symptoms (LUTSs) and undergoing transrectal ultrasound (TRUS)-guided prostate biopsy based on either elevated serum prostate-specific antigen (PSA) (>4.0 ng/ml) or suspicious digital rectal examination (DRE). The performance of miRNAs 21 and 221 in differentiating prostate cancer from nonmalignant cases was evaluated and compared to DRE and elevated PSA. miRNA 21 was overexpressed in 90 % of group A vs. 10 % of group B, while miRNA 221 was overexpressed in 80 % of group A vs. 20 % of group B (p = 0.001). MiRNA 21 overexpression had the highest performance as a diagnostic test with a sensitivity of 90 % and a specificity 90 % (p = 0.02). No correlations were noted between Gleason score of prostate cancer cases and relative quantity (RQ) 21 (r = -0.355, p = 0.292) or RQ 221 (r = -0.044, p = 0.892). Our study showed that serum miRNAs 21 and 221 expression profiling tests may be used as specific noninvasive molecular biomarkers for prostate cancer diagnosis due to their higher sensitivity and specificity with a high negative predictive value leading to a decrease in the biopsies taken for patients with elevated serum PSA values.