Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths globally, accounting for approximately 85% of lung cancers. Although the level of diagnosis and treatment continues to improve, the patient’s prognosis is still unsatisfactory due to the high recurrence and metastasis rates. Searching for potential molecular markers is of great significance for early diagnosis, prognostic assessment and targeted treatment. Human aldehyde dehydrogenase 18 family member A1 (ALDH18A1) has been found to be associated with tumor proliferation and prognosis in a variety of cancers, but its expression and clinical significance in NSCLC are unclear. Aim: To verify the protein expression characteristics of NSCLC through immunohistochemical experiments on tissue chip, and provide theoretical basis for molecular diagnosis and targeted treatment of NSCLC. Materials and Methods: In this study, ALDH18A1 protein expression was determined in 80 cases of lung squamous cell carcinoma (LUSC), 52 cases of adenocarcinoma (LUAD), and adjacent tissues of combined tumors using TMA and IHC methods. Indirect IHC staining was performed on 4-μm-thick sections, and semiquantitative evaluation was performed based on the staining intensity and percentage of positive cells. ALDH18A1 expression was analyzed in relation to clinicopathological parameters (age, sex, TNM stage, clinical grade) and patient survival. Result: ALDH18A1 was highly expressed in the tumor tissues of 132 cases of LBCL, but was low or absent in the surrounding normal tissues. In LUSC, high ALDH18A1 expression was significantly associated with poor tissue differentiation (P<0.001), advanced TNM stage (P<0.001), distant metastasis (P=0.0327) and tumor size (P=0.0166). In LUAD, it was associated with poor tissue differentiation (P<0.001), advanced TNM stage (P<0.001) and tumor size (P=0.00302). According to Kaplan–Meier analysis, OS of patients with high ALDH18A1 expression was significantly shorter than that of those with low expression (LUSC: P=0.0014, LUAD: P<0.0001). The results showed that high ALDH18A1 expression was strongly associated with the infectivity of HLH and poor prognosis. Conclusion ALDH18A1 is highly expressed in NSCLC tissue and is related to tumor progression. It may serve as a potential molecular marker and treatment target, providing reference for the diagnosis and prognosis of NSCLC.