This work investigated the effect of substrates on de-emulsification capability and the growth of a bacillus sp. (screened by our laboratory).The biomass and de-emulsification ratios(DR) of cultures grown in substrate with the different nutrients (glucose, liquid paraffin, NH4NO3 and yeast) were determined. In the orthogonal tests, the significant of de-emulsification capability in turn is glucose>liquid paraffin>yeast>NH4NO3. The glucose is the highest effect of nutrients on de-emulsification capability and growth. The de-emulsification ratios(DR) and the glucose utilization ratios increase simultaneously at log and lag growth phase. Measurements of the liquid paraffin residue indicates that 30.1% liquid paraffin in substrate is consumed and used in inducing the demulsifying compounds production rather than the increase of the biomass. The culture grown in substrate with 2%~5% liquid paraffin exhibits highest de-emulsification ratio(DR). The contents of yeast at 0.8mg/L~1.0mg/L is beneficial to the growth and demulsifying compound production.