The identification of reliable molecular biomarkers in prostate cancer early diagnosis is clinically desirable. We quantitatively detected prostate cancer specifically overexpressed genes, DD3 and PSA, in urine sediments of men suffering from prostate cancer or benign prostate hyperplasia, after prostatic massage. As both genes are exclusively expressed in androgen receptor expressing human prostate carcinoma cell lines, we further investigated the possible effect of androgens on PSA and DD3 gene expression. DD3 and PSA mRNA levels were measured by real-time polymerase chain reaction. The combined markers test had a sensitivity of 80.2% and a specificity of 100%. Both gene transcripts were significantly upregulated by androgens. Results indicated the clinical usefulness of the combination of DD3 and PSA as molecular markers in the early diagnosis of prostate cancer and the need of combining as many as possible analytical data with the clinical and demographic ones to achieve the maximum level of diagnostic accuracy.