Abstract: Objective：To explore the clinical efficacy of biportal video-assisted thoracoscopic surgery (VATS) in the treatment of non-small cell lung cancer. Methods：A total of 536 patients with non-small cell lung cancer receiving surgical resection from January 2013 to December 2016 were enrolled. The surgery was performed as video-assisted thoracoscopic surgery, by which 3-portal VATS for 167 patients (3-portal group) and biportal VATS for remaining 369 patients (biportal group). The clinical effect in two groups was compared. Results：The statistical significances of following parameters were not found in two groups including operation time, intraoperative blood lose, lymph node dissection number, the intraoperative conversion rate, drainage on 1st day after surgery, duration of chest drainage, duration of hospital stay and incidence of postoperative complications. However, compared with 3-portal group, visual analogue scores (VAS) on 1st day, 5th day and 30th day in biportal group were significantly lower. Conclusions：There is no significant difference between biportal VATS and 3-portal VATS in safety and effectiveness. Biportal VATS is a better recommended skill because of less postoperative discomforts.