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One practical method to increase the density of Habrotrocha sp., which is a kind of useful bdelloid rotifer in the wastewater biological treatment system, is to cultivate the metazoa in certain numbers and then put it into the treatment system. Therefore, it is necessary to develop a method which can efficiently cultivate Habrotrocha sp.. In this paper, the optimization of culture condition for Habrotrocha sp. including food dosage, temperature, salinity and pH was investigated by using response surface method. Flour was used as the food of Habrotrocha sp. based on our previous work. The results showed the optimum food particle concentration, temperature, salinity and pH value for culturing Habrotrocha sp. were 7.6×106 particals/mL, 28.2°C, 187mg/L and 6.3, respectively. The actual population of Habrotrocha sp. cultured under the optimized culture condition was 184.0 ind/mL, which is almost the same as the result predicted by response surface method.