Abstract: Objective To evaluate the efficacy and safety of second-line chemotherapy combined with immunotherapy versus second-line single chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). Methods A total of 53 NSCLC patients, who had progressed after first-line chemotherapy and were admitted to the First People's Hospital of Yancheng from June 2019 to October 2021, were selected. Among them, 30 patients received second-line single chemotherapy (a single chemotherapy group), and 23 patients underwent second-line chemotherapy combined with immunotherapy (a combined immunotherapy group). Their efficacy were evaluated and the objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were calculated. Adverse reactions in the two groups during second-line treatment of NSCLC were recorded. Results In the combined immunotherapy group, there was 1 case of complete response (CR), 12 cases of partial response (PR), 4 cases of stable disease (SD), and 6 cases of progressive disease (PD), while the ORR was 56.5% (13/23), and the disease control rate (DCR) was 73.9% (17/23). In the single chemotherapy group, there were no cases of CR, 10 cases of PR, 5 cases of SD, and 15 cases of PD, while the ORR was 33.3% (10/30), and the DCR was 50.0% (15/30). There was no statistical difference in ORR and DCR between the two groups (P=0.091 and P=0.078). The median PFS was 5.5 months for the single chemotherapy group, and 7.0 months for the combined immunotherapy group, with statistical differences (P=0.004). Patients with TNM stages Ⅲ and Ⅳ lymph node metastasis benefited more in the combined immunotheray (P=0.010). The median OS was 10.0 months for the single chemotherapy group, and 11.0 months for the combined immunotherapy group, with statistical differences (P=0.012). PD-L1 positive patients had more benefits in the combined immunotherapy (P=0.038). The incidence of adverse reactions in the combined immunotherapy group did not statistically increase (P>0.05). Conclusions Compared with chemotherapy alone, second-line chemotherapy combined with immunotherapy in NSCLC patients shows no statistical differences in ORR and DCR, but the long-term efficacy is significantly improved.