To study the formation mechanism and the chemical component of biofouling, the biofouling formation was simulated by the dynamic simulation device of circulating cooling water under the constant condition of 30°C and 0.4 m/s. The slime-forming bacteria and Iron Bacteria were selected as the research subject. Adsorption theory of bacteria in the solid/liquid interface was analyzed by colloidal stability theory. The two kinds of bacteria on heat transfer and the relationship between the total number of bacteria and fouling resistance were studied. The main component of biofouling was characterized using XPS. The results show that Iron Bacteria on the negative effects of heat transfer is greater than the slime-forming bacteria. The total number of bacteria is the maximum in the end of the induction period. The main component of biofouling of the slime-forming bacteria of iron bacteria is C, O, N, Fe, Ca, Mg, etc. The major elements of the ratio in different strains exists diversity in the stainless steel heat exchanger, but the most important elements is carbon and oxygen.