Abstract: Bacillus subtilis Tpb55, an antagonistic bacteria isolated from the phyllosphere of tobacco, strongly inhibits the growth of Alternaria alternata (Fries) Keissler, which causes the tobacco brown spot. Crude extract was obtained by precipitation of the cell-free culture of Tpb55 with 90％ saturated (NH4)2SO4. The suspension of the precipitate strongly inbibits Alternaria alternata (Fries) Keissler, and it is thermostable, resistant to trypsin, proteinase K, pepsin and organic solvent. Its active pH range is wide, from 5.0 to more than 10.0. One antagonistic peak was obtained from the crude extract of Tpb55 after DEAE-sepharose Fast Flow and Sephadex G-75, which was shoed only single band with 47.8 kD and PI7.2 in SDS-PAGE, respectively. Analyzed the N-terminal partial sequence of protein was Met-Glu-Ile-Phe-Lys-Tyr-Met-Glu-Thr-Tyr-Asp-Tyr-Glu-Gln-Leu-Val-Phe-Cys-.