In the progresses of prostate cancer (PCa), sarcosine levels increase not only in cells but also in urine samples, suggesting that the measurement of sarcosine in urine can benefit in the creation of non-invasive diagnostic methods for PCa. In this work, a biosensor has been developed for the quantification of sarcosine via the cross coupled chemical and electrochemical reactions with a limit of detection (LOD) of 0.4 nM (S/N = 3). The biosensor has been successfully applied to the analysis of sarcosine in urine samples, which has a great potential application in noninvasive clinical diagnosis of PCa.