Objective: To analyze the imbalance of intestinal flora and the risk factors of incision infection in patients after gastrointestinal surgery. Methods: A total of 455 patients who underwent gastrointestinal surgery in North Jiangsu People’s Hospital from February 2020 to February 2022 were retrospectively analyzed. Thirty-five patients with postoperative incision infection were selected as an infection group, and 420 patients without postoperative incision infection were selected as a non-infection group. The etiological specimens of patients with postoperative incision infection were sent for inspection to detect the distribution of pathogenic bacteria and the distribution of intestinal flora in the 2 groups. The risk factors of incision infection in patients after gastrointestinal surgery were analyzed by univariate and multivariate analysis. Results: The incidence of incision infection in patients after gastrointestinal surgery was 7.69%. The number of Escherichia coli and Enterobacter in the infection group was higher than that in the non-infection group, and the number of Bifidobacteria was lower than that in the non-infection group (all P<0.05). In the infection group, the postoperative intestinal infection proportion after the surgery with age ≥65 years old, emergency surgery, surgical incision ≥10 cm, grade III/IV incision, open surgery, general operating room, number of visitors to the operation >3, operation time >2 h, the non-first operation, was higher than that in the non-infection group (all P<0.05). Multivariate logistic regression analysis showed age, operation timing, length of incision, grade of incision, operation method, the type of operating room, the number of visitors to the operation, and the non-first operation were the gastrointestinal independent risk factors for intestinal infection in postoperative patients (all P<0.05). Conclusion: Patients after gastrointestinal surgery have a high risk of infection, and may have an imbalance of intestinal flora. The pathogenic bacteria are mainly Gram-negative bacteria. The risk factors are clear. Clinical measures should be actively taken to improve the prognosis.