【Abstract】 Objective To investigate the clinical value of serum thymidine kinase 1 (TK1), cytokeratin 19 fragment 21-1 (CYFRA21-1) and squamous cell carcinoma antigen (SCC-Ag) combined with chest CT imaging features in the diagnosis of benign and malignant pulmonary nodules. Methods 105 patients with pulmonary nodules who were treated in the hospital from February 2017 to January 2020 were selected as the research subjects. All of them were subjected to chest CT examination and detection of serum TK1, CYFRA21-1 and SCC-Ag levels. According to pathological findings, the patients were divided into benign group and malignant group. Serum TK1, CYFRA21-1 and SCC-Ag levels and chest CT imaging features of the two groups were compared. The diagnostic efficiency of serum TK1, CYFRA21-1 and SCC-Ag combined with chest CT for benign and malignant pulmonary nodules was analyzed. Results Serum TK1, CYFRA21-1 and SCC-Ag levels in the malignant group were higher than those in the benign group (P＜0.05). The proportions of lobulation sign, spicule sign, pleural indentation and vessel convergence sign in the malignant group were higher than those in the benign group (P＜0.05). ROC curve analysis showed that the AUC value of serum TK1, CYFRA21-1 and SCC-Ag combined with chest CT lobulation sign, spicule sign, pleural indentation and vessel convergence sign in differential diagnosis of benign and malignant pulmonary nodules was the largest (0.901). Meanwhile, the diagnostic sensitivity and specificity were the highest (86-67% and 88-89%). Conclusion Patients with malignant pulmonary nodules have higher serum TK1, CYFRA21-1 and SCC-Ag levels. Besides, chest CT imaging features mainly include lobulation sign, spicule sign, pleural indentation and vessel convergence sign. Serum TK1, CYFRA21-1 and SCC-Ag combined with chest CT imaging features have certain value in the differential diagnosis of benign and malignant pulmonary nodules.