To better separate biological sulfur from denitrifying sulfide removal (DSR) process, we determined a new method by researching on the distribution characteristics of biological sulfur in this process system, Zeta potential, particle size distribution, scanning electron microscope and energy spectrum analysis. The results showed that the biological sulfur in the effluent and sludge accounted for 65％ and 35％ of the total sulfur production, respectively. S content of biological sulfur particles is no less than 60％. Zeta potential value is approximately -20 mV (between ±30 mV); Biological sulfur particles are spherical with rough surfaces, with many functional groups on the surface such as polysaccharide and proteins. The above biological properties indicate that it has characteristics of colloid. The biological sulfur flocculation rate is approximately 99％ when using cationic coagulant to separate biological sulfur. Coagulation is an effective method of separating biological sulfur form (DSR) process.