Hsp90-beta was investigated as prognostic factor because of its apparent association with tumorigenesis. The aim of this study was to investigate the expression of Hsp90-beta in lung cancer patients, to analyze the relationship with respect to the clinicopathological features and to assess whether Hsp90-beta as a potential serum marker for lung cancer. Expression of Hsp90-beta was examined using immunohistochemistry, in-situ hybridization, western blot and enzyme-linked immunosorbent assay. Sensitivities and specificities for Hsp90-beta serum test were determined using receiver operator characteristic curve and cutoff was defined based on 95% and 85% sensitivities. Lung cancer tissues exhibited higher expression of Hsp90-beta than the normal tissues (P< 0.05) and the serum Hsp90-beta of lung cancer patients also exhibited higher level than control groups (P< 0.05). Moreover, increased serum Hsp90-beta was significantly associated with the pathological grade and clinical stage of lung cancer patients (P< 0.05). Using receiver operator characteristic curve analysis, the cutoffs for distinguishing lung cancer from normal and benign groups were 1.155 and 1.158 ng/ml respectively. The sensitivities of Hsp90-beta for distinguishing lung cancer from normal and benign groups were 98.77% and 95.9%, and specificities were 88.33% and 72.7%. Up-regulation of serum Hsp90-beta was associated with pathological grade and clinical stage of lung cancer patients, which indicated that it could be considered molecular biomarker for diagnosis and prognosis of lung cancer.