Objective To find humoral protein markers to develop new experimental diagnostic methods for tuberculous pleurisy. Methods Proteomes of 7 d and 14 d culture filtrate of Mycobacterium tuberculosis growing in Middlebrook 7H9, serum and pleural effusion from five patients with tuberculous pleurisy were detected by surface-enhanced laser desorptionionization time-of-flight massspectrometry (SELDI-TOF-MS). And the data were analyzed with descriptive statistics method to observed the protein components all owned by three kinds of proteome. Results From protein species, proteins of all culture filtrate were far more than that of pleural effusion and serum while proteins of pleural effusion in four cases were more than that of serum. The kinds of common proteins between culture filtrate and pleural effusion, between culture filtrate and serum, between serum and pleural effusion, among culture filtrate and pleural effusion and serum were different. But the protein of relative molecular mass of 2 660 depending on the ratio of mass to charge existed in all samples of culture filtrate, pleural effusion and serum. Conclusion The protein of relative molecular mass of 2 660 possess the latent quality as a specific humoral protein marker for tuberculous pleurisy. But it is essential that must be further confirmed among large samples.