Objective: To investigate the effect of methylene blue tracing on the effect of surgical resection and the prognosis of gastric cancer patients in D2 radical surgery under laparoscope. Methods: In this retrospective cohort study, 160 patients with advanced gastric cancer who underwent surgical treatment in Xinxiang Central Hospital, the 4th Clinical College of Xinxiang Medical College from January 2016 to January 2019 were selected for retrospective analysis. Among them, 84 patients underwent laparoscopic D2 radical gastrectomy for gastric cancer combined with methylene blue labeling operation (labeling group), and the other 76 patients underwent only laparoscopic D2 radical gastrectomy for gastric cancer (control group). The difference of intraoperative and postoperative recovery, lymph node dissection, and postoperative 3-year cumulative survival rate between the two groups were analyzed. Results: The age of patients in the labeled group and the control group were (64.9±7.8) and (66.0±8.3) years old, respectively (P=0.389); And the male patients accounted for 61.9% (52 cases) and 55.3% (42 cases), respectively (P=0.394); The operation time in the labeled group was (218.5±19.6) min, which was shorter than that in the control group (230.1±17.4) min (P<0.001). There was no significant difference between the labeled group and the control group in the amount of bleeding during operation, the time of anal exhaust after operation, the time of eating after operation, the time of hospitalization after operation, and the average diameter of lymph nodes (P>0.05). The total number of dissected lymph nodes, D1 lymph nodes and D2 lymph nodes in the labeled group were significantly higher than those in the control group (all P values<0.05). The operative complication rate in the labeled group was 11.9% (10 cases), which was lower than that in the control group (25.0%, 19 cases) (P=0.032); There was no statistical significance in 3-year cumulative survival rates of patients between the labeled group (61.9%) and the control group (52.6%) (χ2=3.46,P=0.065). Conclusion: The use of methylene blue tracing in laparoscopic D2 radical surgery for gastric cancer is beneficial to reduce the operation time, improve the lymph node clearance rate, and reduce surgical complications.