Prostate cancer (PCa) is one of the most common cancers among men. Presently, a screening test for PCa is based on serum prostate specific antigen (PSA) level. However, PSA level is not a specific biomarker for PCa. Recently, several potential biomarkers of PCa have been discovered. In this study, detection methods for sarcosine and PCA3 in urine have been developed for PCa screening and diagnosis. An enzyme-coupled colorimetric method was proposed for sarcosine assay. This method is a specific reaction based on sarcosine oxidase (SOX) and horseradish peroxidase (HRP). The method was performed in the 96-well microtiter plate and measured spectrophotometrically (OD570). At the optimum conditions, the limit of detection (LOD) and quantification (LOQ) were found to be 0.7 and 1 μM, respectively. A good linearity was revealed with a coefficient of 0.990. The assay showed no significant interference from ascorbic acid, glucose and bilirubin. In addition, it is extremely specific for sarcosine rather than other amino acids. The determination of sarcosine in human urine displayed high accuracy and good reproducibility. This method is promising to differentiate among prostate cancer patients, benign prostatic hyperplasia patients and healthy subjects according to urinary sarcosine level. Simultaneously, a gold nanoparticle-based colorimetric assay was also achieved for PCA3 detection. The assay used the property of thiol group on the 5’-end of the PCR product to simply immobilize onto the surface of gold nanoparticles (AuNPs), which can be able to prevent self-aggregation of AuNPs under high concentration of NaCl solution. Our assay can distinguish the positive from negative results by the naked eye observation of color change or using the ratio of the absorbance at 520 and 640 nm (A520/640). This method was also performed in the 96-well microtiter plate. At the optimum conditions, the optimal ratio (520/640) was > 1.5 in the presence of thiolated PCR product. The sensitivity of assay was approximately 31.25 ng of PCR product. In addition, the assay showed significantly specific for prostate cancer cell lines, which PCA3 is highly expressed, while the other cell types showed no PCR product. Altogether, this study provides the rapid, simple and specific tools to determine urinary sarcosine and PCA3 which could be useful for prostate cancer screening and diagnosis.