Objective To analyze the expression levels of serum miR-21-5p and miR-100-5p in patients with esophageal squamous cell carcinoma(ESCC)and healthy controls,and to explore their impact on the differential diagnosis,clinical characteristics correlation,and patient prognosis of ESCC.Methods A total of 158 patients with advanced ESCC who underwent radiotherapy in our hospital's radiotherapy department from January 2018 to January 2022 were collected as the experimental group.Additionally,150 healthy individuals who underwent physical examinations were selected as the control group.Peripheral blood(5 ml)was collected from each subject,and serum was prepared.The expression levels of miR-21-5p and miR-100-5p were detected using real-time quantitative PCR(RT-qPCR).The expression differences of miR-21-5p and miR-100-5p between the experimental and control groups were compared using the t-test,and the clinical prognosis survival analysis was performed using the Kaplan-Meier method with Log-Rank test.Results Compared with the healthy control group,the expression levels of serum miR-21-5p and miR-100-5p in ESCC patients were significantly increased(both P＜0.001).The elevated expression levels of serum miR-21-5p and miR-100-5p can accurately distinguish ESCC from healthy controls,with an area under the curve(A UC)of 0.918 for combined diagnosis,a sensitivity of 93.2％,and a specificity of 87.1％.The increased expression of serum miR-21-5p and miR-100-5p in ESCC patients was correlated with later TNM stages and poorer disease-free survival and overall survival(both P＜0.05).Multivariate Cox analysis showed that TNM stage Ⅲ,high expression of serum miR-21-5p,and low expression of miR-100-5p are independent risk factors for the disease-free survival and overall survival of ESCC patients(all P＜0.05).Conclusion The expression levels of serum miR-21-5p and miR-100-5p are significantly increased in ESCC patients and indicate a poorer prognosis.Serum miR-21-5p and miR-100-5p hold promise as biomarkers for the diagnosis and prognostic prediction of ESCC.