Objective To investigate the correlation between serum laten transforming growth factor binding protein 2 (LTBP2), type Ⅳ collagen α3 (COL4A3), carbohydrate antigen 125 (CA125) levels and clinicopathological features in patients with non-small cell lung cancer (NSCLC) and their diagnostic value. Methods A total of 97 patients with NSCLC admitted to Zhoukou Central Hospital from May 2021 to May 2023 were included in the observation group, and 97 patients with benign lung lesions were included in the control group. The serum levels of LTBP2 and COL4A3 were measured by double antibody enzyme-linked immunosorbent assay, and the serum levels of CA125 were measured by double antibody sandwich method. The serum levels of the two groups were compared, and the correlation between the levels of each index and pathological characteristics and the diagnostic value of combined detection for NSCLC were analyzed. The correlation between each index level and pathological characteristics was analyzed, as well as the diagnostic value of combined detection in NSCLC. Results Serum LTBP2, COL4A3 and CA125 levels in observation group were higher than those in control group (P<0.05). Serum levels of LTBP2, COL4A3 and CA125 in patients with different TNM stages, depth of invasion and lymph node metastasis were compared: Ⅲ~Ⅳ>Ⅰ~Ⅱ, T3+T4 > T1+T2, with lymph node metastasis > without lymph node metastasis (P<0.05). Serum levels of LTBP2, COL4A3 and CA125 were positively correlated with TNM stage, invasion depth and lymph node metastasis in NSCLC patients. The AUC of all indexes in the combined diagnosis of NSCLC was 0.889, the best sensitivity and specificity were 90.72% and 84.54%, respectively, and the Yoden index was 0.753. Conclusion Serum LTBP2, COL4A3 and CA125 levels are positively correlated with clinicopathological features of patients with NSCLC. Combined detection has certain diagnostic value for NSCLC, and can be used as auxiliary indicators for clinical diagnosis of NSCLC.