We aimed to explore the relationship between the circadian gene TIMELESS (TIM) in non-small-cell lung cancer (NSCLC) and prognosis, so as to provide a reference for subsequent targeted therapy and patient prognosis. A total of 72 patients with NSCLC were selected as the study cohort. Immunohistochemistry and Western blot were used to measure the expressions of TIM in NSCLC and para-carcinoma tissues (PCT). RT-qPCR was used to measure the mRNA expression of TIM. The Kaplan-Meier method was further utilized to generate survival curves for a log-rank test single-factor analysis and a Cox regression multi-factor analysis. The relationship between the TIM gene expression and the clinical features and survival were explored. The results indicated that the mRNA and protein expressions of TIM in the NSCLC tissues were higher than those in the PCT ( P < 0.01). An immunohistochemistry assay showed that the expression rate of TIM in the NSCLC tissues of the 72 cases was 84.72%. The TIM expression was significantly different in terms of the NSCLC differentiation level, the tumor size, the lymph node metastasis, the TNM stage, and the survival rate (P < 0.05, P < 0.01). A Cox regression analysis indicated that the degree of TIM expression can be used as an independent observation index that indicates the prognosis of NSCLC. The TIM expression was significantly increased in the NSCLC tissues, and the higher the degree of positive expression, the worse the clinical prognosis. The expression of TIM is of certain auxiliary value in determining the degree of NSCLC malignancy, the lymph node metastasis, and the evaluation of the prognosis.