Abstract: The biological slime was developed with adding different concentration oil into the circulating cooling water to simulate medium leaking phenomenon in oil refining enterprise. The water quality of the circulating cooling water and biofouling characteristics were investigated under the influence of medium leaking, and microstructure of biological slime was observed by using scanning electron microscope (SEM). The results show that the content of turbidity, COD, petroleum hydrocarbon, total iron and zinc ion are widely changed with variation of diesel dosage, but the total hardness and total phosphorus has little change. The content of biomass, polysaccharide, protein content, EPS and fat phosphorus have obvious volcanic type change trends as with the diesel dosage increasing. Combined with the results of the SEM, it can be drawn that medium leaking has positive effect on the biological slime at the diesel dosage of 0.1，0.2，0.3，0.5 g/L, but negative effect on the biological slime will occur at the diesel dosage of 0.7, 0.9 g/L. These results can be very help to select biocides for oil refining enterprise.