Tuberculosis (TB) is still one of the leading causes of death worldwide. Due to the ever-growing problem of Mycobacterium tuberculosis (Mtb) drug resistance, TB is still not well controlled. There is an urgent need to develop new technology for promoting Mtb basic research, and to reveal new mechanisms, identify new targets, and develop new anti-Mtb drugs. Bore this need in mind, a Mtb functional proteome microarray was constructed to promote basic research. Considering the high virulence of Mtb, long replication cycle, it is very difficult to perform Mtb related studies, thus efficient tool is need. The proteome microarray is a powerful tool for the researchers in the field of TB study. It has already been successfully applied in a wide range of studies. Better understandings of a variety of key biological processes have already been added, for example, Mtb-host interactions, small-molecule-protein binding and mechanism of antibiotic resistance. In order to help readers to better understand this powerful tool, we present several major applications of the Mtb functional proteome microarray in this review, we anticipate researchers will adopt this powerful tool for a variety of TB related studies, thus facilitate both basic and clinical research of TB.