[Objective] To establish and apply an high-throughput assay for screening inhibitors of Mycobacterium tuberculosis protein kinase B (PknB). [Methods] The kinase domain of Mycobacterium tuberculosis PknB was first cloned and expressed in Escherichia coli. The purified protein is utilized for a high-throughput assay for screening PknB inhibitor. [Results] Eight out of 18 000 compounds were identified as exhibiting inhibitory effect upon PknB activity using the screening assay, among which three compounds showed antibacterial activity against Mycobacterium tuberculosis, Mycobacterium marinum and Mycobacterium smegmatis. [Conclusion] The high-throughput screening model has advantages of high sensitivity and stability, which can be used in anti-TB drug screening. Three compounds are worth further study because of their great activities against both PknB and bacterial growth.