Surface proteins consist of secreted and membrane proteins and play a central role in the interaction of the pathogen with its environment, especially in the pathogenicity of Mycobacterium tuberculosis (MTB). Research on surface proteins in MTB has focused on 2D electrophoresis of culture filtrate proteins (CFP), extraction of transmembrane proteins with detergent and predicting their properties with a range of available algorithms. However, functional analysis of these secretomes is possible only if many proteins are expressed and purified individually, which limits a large number of studies to the function of the proteome. Here, we utilized a phage display system to construct a whole genomic surface protein phage display library of MTB, which can complete direct selection, identification, expression, purification and functional research of surface proteins of MTB. With this system we made a new serological approach involving iterative subtraction screening. Cross-reactivity of antibodies was reduced by preadsorption of the surface protein phage display library with the sera of healthy BCG-vaccinated individuals prior to studying their reactivity against the sera of tuberculosis (TB) patients. As a result six antigens were identified, three of which have not previously been reported as diagnosis antigens. The surface protein phage display library shows great promise in the study of MTB.