Three strains (CPW-1, CPW-2 and CWH-1) of tobacco black root rot were isolated from diseased tobacco plant roots, and the biological characteristics of CPW-1 and CWH-1 were studied. The results showed that the optimal culture mediums for CPW-1 and CWH-1 were PSA and PCA, respectively. The two strans could make good use of carbon sources, such as lactose, maltose, starch, glycerine, fructose, and nitrogen sources, such as beef extract and casein. The optimum temperature for mycelia growth was 25°. The optimum pH values for mycelia growth of CPW-1 and CWH-1 were pH7 and pH10, respectively. Under the same temperature and light intensity, the germination rate of CPW-1 conidiospore was higher than that of CWH-1, while which was on the contrary at the presence of organic nutrition. It was favorable for the conidiospore germination in the dark, the optimum temperatures for CPW-1 and CWH-1 conidiospore germination were 20° and 15°, respectively. The percentage of conidiospore germination was the highest in starch solution at pH4.