Abstract: Objective: To compare the difference in mitochondrial DNA (mtDNA) copy number in esophageal squamous cell carcinoma (ESCC), paraneoplastic tissue and normal mucous membrane of the esophagus and to study the relationship between the mtDNA copy number and the occurrence and development of esophageal squamous cell carcinoma. Methods: The mtDNA copy number of 42 specimens of ESCC, paraneoplastic mucous tissue and normal mucous membrane of the esophagus was determined using real-time fluorescent quantitative PCR. Qualitative tests of the mtDNA were conducted using agarose gel electrophoresis. Results: The rate of successful mtDNA amplification was 100% (42/42) for ESCC, paraneoplastic tissue and normal mucous membrane of the esophagus, with the average mtDNA copy number detected at 27.189 4× 106μ g DNA, 9.410 2× 106μ g DNA and 5.934 7×106μ g DNA, respectively. There were significant differences (F=27.83,P<0.05) in mtDNA copy number among the three. A strong band was detected at 403bp after gel electrophoresis of the PCR products, and the track where the amplicons from the ESCC tissues were loaded was rather bright, which was in accordance with the results of real-time PCR. Conclusion: Incremental increases in the mtDNA copy number is related to occurrence and development of ESCC.