Tobacco black shank is one of the most serious soil-borne diseases in tobacco production. Biological control has been proved effective and environment-friendly, but screening of antagonists against pathogens is the basic work in bio-controlling. Twelve strains of bacteria antagonist against Phytophthora parasitica var．nicotianae were isolated by using the plate-confrontation method from rhizosphere soils under healthy tobacco plants in tobacco fields seriously infected with the pathogen. The averaged inhabiting rates of the antagonists ranged from 59.4% to 71.1%. The best performer Strain C-5, which was identified as Paenibacillus polymyxa (C-5) based on biochemical tests and 16S rRNA sequence similarity, was then selected for further experiments. Results from antibiotic spectrum experiments show that Strain C-5 was also effective in inhibiting other plant pathogen such as Fusarium osyporunm f. sp melonis , Fusarium osyporum f. sp cucumerinum and P. capsici. Pot experiments show that application of bio-organic fertilizer supplemented with Strain C-5 suppressed the incidence of tobacco black shank by 80% as compared with the control. To our knowledge, this is the first report on the microbiological antagonism of Strain Paenibacillus against Phytophthora parasitica var. nicotianae.