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In this study, the optimal conditions of production of bioflocculant by the culture of Pseudomonas fluorescens C-2 were examined, using molasses wastewater to replace glucose as carbon source and energy source in the culture medium. Results showed that the COD concentration in molasses wastewater favorable for the production of the bioflocculant PS-2 was 8000 mgldrL-1, and an optimal culture condition of inoculum size of 5%(v/v), 30degC, initial pH 8.0 and shaking speed of 160 r-min-1, under the optimal culture conditions, the highest flocculating activity achieved for Kaolin suspension was 92.5% and 2.52 g pure PS-2 per liter broth was obtained after 72 h cultivation. The wastewater treatment experiments results indicated the chromaticity removal efficiencies of the bioflocculant PS-2 for breed aquatics wastewater and domestic sewage were above 9% with a PS-2 dosage of 120 mLldrL-1, the turbidity removal efficiencies of the PS-2 for four wastewaters were all above 90% with a bioflocculant PS-2 dosage of 120 mLldrL-1. The bioflocculant PS-2 produced using molasses wastewater substrate possesses flocculating activity comparable or superior to that of bioflocculant produced using conventional substrate.