Lung cancer is one of the most common malignant neoplasms worldwide. CD24 is a marker of tumor stem cells that plays an important role in tumorigenesis. Hsp70 is an important molecular chaperone. However, the co-expression and interaction of CD24 and Hsp70, as well as the significance for the prognosis of lung cancer are still unclear. The expression levels of CD24 and Hsp70 were detected by immunohistochemistry and their correlation was analyzed. The expression levels of CD24 mRNA and protein were examined using qRT-PCR and western blotting in SPCA1, A549, H1975, and H1650 cell lines. A CD24-overexpressing cell model was established. The interaction between CD24 and Hsp70 was verified by co-immunoprecipitation and western blotting. CD24 and Hsp70 expression were significantly higher in lung cancer tissues than in adjacent tissues (CD24: p=0.008; Hsp70: p<0.001). CD24 protein expression showed a positive correlation with lymph node metastasis, TNM stage, and vascular cancer thrombus. Hsp70 protein expression showed a positive correlation with differentiation, lymph node metastasis, and TNM stage. CD24 and Hsp70 high expression were also correlated with poor survival. The positive co-expression rate of CD24 and Hsp70 in lung cancer tissues was 52.7% (49/93). CD24 and Hsp70 expression in lung cancer were positively correlated (r=0.368, p<0.001), and co-immunoprecipitation was verified that both endogenous and exogenous CD24 co-precipitated with Hsp70 directly or indirectly. When Hsp70 inhibitor VER15508 was added to A549 cells, Hsp70 and CD24 protein expression were significantly decreased. The present study demonstrated that CD24 and Hsp70 were highly expressed in lung cancer tissues, and associated with invasion, metastasis, and poor survival. Hsp70 may regulate CD24 expression. Co-expression of CD24 and Hsp70 may be a prognostic biomarker for lung cancer.