Prostate cancer is a kind of commonly diagnosed male malignancy. With the aging population in China, both incidence and mortality of prostate cancer are expected to keep increasing in the future. The methylation of RARβ2 gene promoter is a common molecular event in prostate cancer. Thus, we aimed at establishing a high-performance noninvasive DNA methylation assay based on pyrosequencing for screening of prostate cancer in this article. The assay is designed to detect aberrant promoter methylation of RARβ2 gene in ejaculate samples. The negative and positive control plasmids were constructed with different treatments by direct bisulfite conversion or conversion after Sss I Methylase methylation to establish quality control standard. The ejaculate and tissue samples were collected from patients with histologically confirmed adenocarcinoma of prostate (n = 43) and benign prostatic hyperplasia (n = 40). Significant correlation was observed between prostate cancer and methylation level of RARβ2 gene promoter. In addition, the results of pyrosequencing in ejaculate samples were compared with that of DNA sequencing in tissue samples from the same patients. There is no significant difference in the detection of RARβ2 promotor methylation between these two methods (p < 0.05). In conclusion, we have developed a high-performance noninvasive DNA methylation assay based on pyrosequencing which is more suitable for high-throughput detection of aberrant promoter methylation in ejaculate samples. Moreover, the acceptive degree of this noninvasive method makes it potentially promising for future screening of prostate cancer.