Objective To compare the efficacy and safety of layered hand ⁃sewn end to end invagination anastomosis versus instrument anastomosis in combined thoracoscopic and laparoscopic definitive resection for esophageal cancer. Methods The present study was designed as a retrospective exploratory study，patients with newly diagnosed esophageal cancer who were admitted to the department from January 1st 2018 to December 31st 2019 were assigned to experimental group（layered hand ⁃ sewn end to end invagination anastomosis）and control group（instrument anastomosis）based on the actual surgical therapeutic options received in clinical practice. The baseline clinical characteristics and perioperative clinical manifestations of the patients were collected and were followed up regularly after hospitalization to obtain the prognostic data of the patients. Regarding the observational endpoints，the differences of perioperative indicators and adverse events between the two groups were compared. The disease⁃free survival（DFS）and overall survival（OS）were compared between the two groups. Results A total of 123 patients with esophageal cancer who underwent surgical resection were enrolled，including 63 patients in the experimental group and 60 patients in the control group. All patients were followed up regularly，the incidence of anastomotic leakage in the experimental group was significantly lower than that of the control group after 36 months （1.6% vs. 10.0%，P = 0.045）. The incidence of anastomotic stenosis in the experimental group was significantly lower than that of the control group after 36 months（6.3% vs. 20.0%，P = 0.024）. Furthermore，the incidence of gastroesophageal reflux in the experimental group was significantly lower than that of the control group（22.2% vs. 41.7%，P = 0.021）. In terms of perioperative safety profile，no significant differences in adverse reactions such as pulmonary infection，incision infection，arrhythmia and pleural effusion between the two groups were noted（P > 0.05）. Prognostic data showed that the median DFS of the experimental group was longer than that of the control group（47.1 months vs. 39.3 months，χ2 = 4.300，P = 0.038）. The median OS of the experimental group was longer than that of the control group（NR vs. 44.1 months，χ2 = 5.733，P = 0.017）. Conclusion Layered hand ⁃ sewn end to end invagination anastomosis combined with thoracoscopic and laparoscopic definitive resection of esophageal cancer might attenuate the incidence of anastomotic leakage，anastomotic stenosis and gastroesophageal reflux with tolerable safety profile. Preliminary prognostic data suggested that it might bring long⁃term survival benefits for the patients. The conclusions should be confirmed in prospective clinical trials subsequently.