Background: To investigate the use of uniportal video-assisted thoracoscopic surgery (U-VATS) in the treatment of non-small cell lung cancer (NSCLC). Materials and Methods: A total of 82 patients with early NSCLC in our hospital from May 2019 to January 2021 were enrolled to this study. Forty-one patients treated with spontaneous breathing U-VATS were the research group (RG), and 41 with conventional thoracoscopy were the control group (CG). Fasting peripheral blood was drawn from patients before (T0), one (T1) and three days after surgery (T2), respectively. White blood cells (WBC), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinase-9 (MMP-9) and T-lymphocyte subsets were measured in the blood. In addition, we counted the conversion rate to open chest and tracheal intubation in the RG, and compared the operation time, intraoperative bleeding, chest drainage, chest tube retention time, antibiotic application time, postoperative hospital stays and complications between groups. Finally, a 1-year prognostic follow-up was performed to record overall survival, chemotherapy rate, and recurrent metastasis rate. Results: At T1, WBC, IL-6, TNF-alpha, and MMP-9 were lower in the RG than in the CG, while immune function was better in the RG at T1 and T2 (P<0.05). Complication rates were lower in the RG than in the CG when compared (P>0.05). The prognosis of 1-year overall survival, chemotherapy rate, and recurrence rate were not different between the two groups (P>0.05). Conclusion: Spontaneous breathing U-VATS can effectively reduce the inflammation and enhance the stability of immune function in NSCLC patients undergoing surgery.