To explore the flora characteristics and differences of esophageal tissues between elderly esophageal squamous cell carcinoma (ESCC) patients and young and middle-aged ESCC patients, so as to assist in studying the potential biomarkers of elderly ESCC patients. In this study, a retrospective study was adopted. 72 ESCC patients diagnosed in Taihe Hospital, Shiyan City, Hubei Province from July 2018 to July 2019 were selected, including 49 patients in the elderly group (≥ 60 years old, 40 males and 9 females), 23 patients in the young and middle-aged group (<60 years old, 21 males and 2 females). In the same period, 20 healthy persons without abnormal gastroscopy in endoscopy center were selected as the control group (aged 35-78 years old, median age 57 years old, 16 males and 4 females). The genomic DNA was extracted from the affected esophageal tissues of patients with ESCC and the middle esophageal samples of the control group. The V4 hypervariable region of bacterial 16SrRNA gene sequence was amplified. Illumina HiSeq sequencing technology was adopted. The flora characteristics of elderly, young and middle-aged ESCC patients was compared and analyzed. QIIME and Rstudio software were used to analyze the sequence data, and nonparametric Kruskal-Wallis test or Wilcoxon rank sum test were used for statistical methods. Shannon index [5.17 (4.53, 5.95) 4.79 (3.74, 5.97)], Simpson index [0.94 (0.91, 0.96) 0.92 (0.83, 0.96)] and Chao1 index [343.55 (259.76, 570.59) 329.16 (268.88, 648.00)] were similar in flora of two groups, and there was no significant difference (=-0.791, -1.057, -0.380, all >0.05). There was no significant difference in β-diversity between the elderly group and the young and middle-aged group (PC1=19.14%, PC2=6.95%, =0.67, =0.42). At the phyla level, the top 5 phyla in abundance were as follows: and in the young and middle-aged group, while the top 5 phyla in abundance were as follows: and in the elderly group; the significant difference between the two groups was (=0.596, 0.05). At the genus level, the top 5 genera in the young and middle-aged group in abundance were as follows: and In the elderly group, and were the top 5 in abundance, and there were significant difference in between the two groups (=0.938, 0.05). PICRUSt function prediction showed that the abundance of and in the elderly group were lower than those in the young and middle-aged group (all =0.734, 0.05). There is no significant difference in α-diversity and β-diversity between elderly ESCC patients and young and middle-aged patients, but the abundance of flora increased.