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One hundred eighty pairs of tissues of esophageal squamous cell carcinoma (ESCC) were tested by the transcriptome sequencing in order to explore etiology factors. The chi‐square test and correlation analysis demonstrated that the relative expression levels of keratin 17 (KRT17) and collagen type I α1 chain (COL1A1) were significantly higher in EC with diabetes. Expression of KRT17 was correlated with blood glucose (r = 0.204, p = 0.001) and tumor size (r = −0.177, p = 0.038) in patients. COL1A1 correlated with age (r = −0.170, p = 0.029) and blood glucose levels (r = 0.190, p = 0.015). Experimental results of qRT‐PCR: KRT17 and COL1A1 genes were highly expressed in ESCC (p < 0.05). When the two genes were used as a combination test, the positive detection rate of EC was 90.6%, and the ROC curve had greater power. The KRT17 and COL1A1 genes had the potential to be biomarkers for the diagnosis of ESCC. [ABSTRACT FROM AUTHOR]