Prostate cancer (PCa) is one of the leading causes of cancer deaths in men. Since there are limited treatment options available for the advanced tumors, there is an urgent need for novel diagnostic tools for PCa. Prostate secretion samples (PSS) from 23 PCa and 25 benign prostate hyperplasia (BPH) patients were obtained from Urology Department of Bagcilar Educational and Research Hospital (Istanbul). MicroRNA (miRNA) profiling of eight PSS (four from BPH, four from PCa patients) was performed using microarray. Four of significantly deregulated miRNAs were further confirmed using quantitative reverse-transcription PCR (qRT-PCR). Statistical analysis was performed using Student's t-test. ROC curves were plotted with SPSS-15.0. In this study, we aimed to identify a miRNA expression signature that could be used to distinguish PCa from BPH. MiRNA profiling of four PCa and four BPH patients with microarray revealed that miR-361-3p, miR-133b and miR-221 were significantly downregulated and miR-203 was upregulated in PSS of PCa patients. Further qRT-PCR analysis confirmed the altered expressions of these four miRNAs in PSS of 23 PCa and 25 BPH patients. Four miRNAs, together and individually have much power (AUC; 0.950) than PSA has (AUC; 0.463) to discriminate PCa from BPH patients. We have shown for the first time in the literature the presence of miRNAs in the PSS. We suggest PSS as a powerful non-invasive source for evaluation of prognosis in PCa, since prostate massages can be easily applied during routine examination. Our results showed that certain differentially expressed miRNAs in PSS could be used as diagnostics markers.