Abstract: Biofouling is one of the most serious problems faced in petroleum industry using refinery wastewater as their coolant fluid. Understanding the contribution of nutrient levels to biofouling control is important requirement for management strategies in the recirculating cooling water system. Thus，an orthogonal experiment was designed with BOD₅ (carbon source)，NH₄⁺-N (nitrogen source)，TP (phosphorus source) as the major influencing factors，and the preponderant microorganisms in biofouling were also identified. Results showed that the most important factor affecting polysaccharide，protein，EPS concentration and wet weight is BOD₅，followed by TP and then NH₄⁺-N. When BOD₅ is 5mg/L，NH₄⁺-N is 10mg/L and TP is 1mg/L in the circulating cooling water system，the biofouling is effectively controlled. In addition，the variation of nutrients concentrations in the recirculating cooling water system results in the change of polysaccharide content，protein content and the types of preponderant microorganisms.