Objective: To investigate the auxiliary diagnostic value of seven tumor-associated autoantibodies (AABs) P53, PGP9.5, SOX2, GAGE7, GBU4-5, MAGEA1 and CAGE in early non-small cell lung cancer (NSCLC). Methods: The case-control study enrolled 195 patients with early NSCLC [71 males and 124 females, aged (55.70±11.78) years old], 114 patients with benign lung disease [44 males and 70 females, aged (52.85±12.31) years old] and 100 healthy subjects [39 males and 61 females, aged (53.62±9.97) years old] from the First Affiliate Hospital of Nanjing Medical University from June 2020 to December 2020. AABs were detected by enzyme-linked-immunosorbent serologic assay (ELISA), and carcinoembryonic antigen (CEA),cytokeratin 19-fragments (CYFRA21-1) and neuron specific enolase (NSE) were detected by electrochemiluminescence. The levels of AABs,CEA,CYFRA21-1 and NSE in the 3 groups were compared. Patients with benign lung diseases and healthy subjects were combined into the control group, and the positive rate of each indicator in the NSCLC group and the control group was compared. The diagnostic efficacy of single and combined tests for NSCLC were obtained using receiver operating characteristic (ROC) curves. Besides, the relationship between the levels of AABs, CEA, CYFRA21-1 and NSE and their clinicopathological features and preoperative imaging parameters in NSCLC patients was assessed. Results: The levels of SOX2 [0.70 (0.10, 2.40) U/mL] and GBU4-5 [1.30 (0.30, 8.90) U/mL] in NSCLC group were higher than those in benign disease group [SOX2: 0.50 (0.10, 1.60) U/mL, GBU4-5: 0.80 (0.10, 2.30) U/mL, Z values were 27.258 and 45.797; P values were all<0.05] and health control group [SOX2: 0.45 (0.10, 1.08) U/mL, GBU4-5: 0.75 (0.20, 1.78) U/mL, Z values were 32.551 and 40.456; P values were all<0.05], and there was no significant difference between benign disease group and health control group (Z values were 5.293 and 5.340, P values were all>0.05). The levels of CEA [1.75 (1.08, 2.72) U/mL] and CYFRA21-1 [1.81 (1.41, 2.36) U/mL] in NSCLC group were higher than those in healthy control group [CEA: 1.22 (0.68, 1.81) U/mL, CYFRA21-1: 1.43 (1.14, 1.74) U/mL, Z values were 64.100 and 37.597; P values were all<0.05], but there was no significant difference between NSCLC group and benign group [CEA: 1.74 (1.01, 2.51) U/mL, CYFRA21-1: 1.82 (1.45, 2.46) U/mL, Z values were 7.275 and 10.621; P values were all>0.05]. The positive rates of P53, SOX2, GAGE7, GBU4-5 and CEA in NSCLC group were higher than those in the control group [P53: 10.3% vs 0.9%, SOX2: 11.3% vs 2.3%, GAGE7: 11.3% vs 0.5%, GBU4-5: 30.1% vs 5.6%, CEA: 9.7% vs 0.9%, χ2 values were 17.420, 13.242, 22.485, 43.211, 16.255, respectively; P values were all<0.05]. The diagnostic efficiency of the combined detection of seven AABs was better than that of single detection. Seven AABs combined with CEA [area under curve (AUC): 0.732, sensitivity: 64.10%] and with CYFRA21-1 (AUC: 0.737, sensitivity: 58.97%) greatly improved the diagnostic efficiency and sensitivity of CEA (AUC: 0.583, sensitivity: 50.77%) and CYFRA21-1 (AUC: 0.552, sensitivity: 44.10%). The levels of SOX2 and CEA in NSCLC patients were correlated with the degree of tumor invasion (H values were 6.436 and 14.071; P values were all<0.05); the levels of GAGE7 and CEA were correlated with the nodule density (H values were 7.643 and 12.268; P values were all<0.05); and the levels of SOX2, GAGE7, CEA and CYFRA21-1 were all correlated with the nodule size (H values were 10.837, 11.528, 31.835, 20.338; P values were all<0.05). Conclusion: The detection of AABs combined with CEA and CYFRA21-1 is helpful for the early auxiliary diagnosis of NSCLC, and plays an important role in prevention and screening for early lung cancer.