Abstract: Objective 　This study was designed to detect the methylation status of the promoter region of p16 gene in DNA extracted serum from esophageal squamous cell carcinoma (SCC), and evaluate the role of p16 gene promoter hypermethylation in esophageal SCC screening and early diagnosis. Methods 　Nested methylation-specific PCR (nMSP) was used to detect p16 promoter hypermethylation in serum DNA from 56 esophageal squamous cell carcinoma (SCC) and cont rol serum samples from 22 healthy individuals. Compared methylation-specific PCR(MSP), nMSP was more sensitive. Results 　Aberrant promoter rmethylation of the p16 gene was found in 34 of 56 serum samples using nMSP, and only 15 cases was detected using MSP. No aberrant promoter methylation was detected in the peripheral serum of the healthy individuals. Conclusion 　The detection of p16 gene promoter hypermethylation in the serum of esophageal SCC patient s can give the useful information for tumor early diagnosis, follow-up study of esophageal SCC patient s. nMSP is a simple, sensitive, and specific method for rapid analysis of the promoter methylation status of many genes.