Abstract: Based on sodium acetate method, the addition of antibiotics could significantly improve the isolation efficiency of B.thuringiensis from soil. B.licheniformis, B.pumilus, B.popilliae could grow slightly (colony was rather small), and other 9 contaminants could not grow when the concentration of sodium penicillin and gentamicin reached 400μg/ml. 4 subspecies out of 29 Bt standard strains were slightly or moderately inhibited, but still could form spores and crystals. The other subspecies could grow and form crystal normally. With this method, 10120 soil samples had been screened and 759 strains of B.thuringiensis had been isolated. The total isolation rate was 7.5%, which was higher than that of sodium-acetate method (3.8%). Strains isolated by sodium acetate antibiotics method were primarily classified as 28 types based on gene combinations, in which 26.2% strains showed indeterminable gene types. The gene combination types of this method were richer than those of sodium acetate method. The strains with same gene types were also much lower than that of sodium acetate method. New genes might exist in the strains with indeterminable gene types which expressed 1 to several crystal proteins with novel molecular weight.