Objective To compare the perioperative results between uniportal and three-portal thoracoscopic lobectomy for non-small cell lung cancer (NSCLC). Methods Electronic databases including PubMed, Web of Science, EMbase, CNKI, Wanfang were systematically searched from the establishment of each database until April 2022. Literature screening, data extraction and bias risk assessment were independently conducted by two researchers. All combined results were performed by RevMan 5.3 and Stata 16.0. The quality of the literature and the risk of bias were evaluated using the Cochrane Bias Risk Assessment Tool. Results Eighteen eligible randomized controlled trials (1 597 patients) were identified eventually, including 800 patients undergoing uniportal thoracoscopic lobectomy and 797 patients undergoing three-portal thoracoscopic lobectomy. Meta-analysis results showed that compared to the three-portal approach, uniportal lobectomy took longer operation time (WMD=7.63, 95%CI 2.36 to 12.91, P=0.005) with less intraoperative blood loss (WMD=–28.81, 95%CI –42.54 to –15.08, P<0.001). Furthermore, patients undergoing uniportal lobectomy achieved lower visual analogue score within 24 hours after the operation (WMD=–1.60, 95%CI –2.26 to –0.94, P<0.001), less volume of drainage after the operation (WMD=–25.30, 95%CI –46.22 to –4.37, P=0.020), as well as shorter drainage duration (WMD=–0.36, 95%CI –0.72 to –0.01, P=0.040). Besides, patients undergoing uniportal lobectomy were also observed with shorter length of hospital stay (WMD=–2.28, 95%CI –2.68 to –1.88, P<0.001) and lower incidence of postoperative complications (RR=0.49, 95%CI 0.38 to 0.63, P<0.001). However, the number of lymph nodes harvested during the operation (WMD=–0.01, 95%CI –0.24 to 0.21, P=0.930) was similar between the two groups. Conclusion Both uniportal and three-portal thoracoscopic lobectomy for NSCLC are safe and feasible. The uniportal approach is superior in reducing short-term postoperative pain, postoperative complications and shortening the length of hospital stay.