To reduce ammonium and nitrite in aquaculture water, an isolate of the denitrifying bacterium Pseudomonas stutzeri, SC221-M, was obtained. The effects of various nitrogen and carbon sources, the ratio of carbon to nitrogen and temperature on bacterial growth, denitrification rates and the expression levels of nirS and nosZ in SC221-M were studied. The following conditions were determined to be optimal for growth and denitrification in SC221-M: NaNO2 as the nitrogen source, sodium citrate as the carbon source, a carbon to nitrogen ratio range of 4-8, and a temperature range of 20-35°C. Subsequently, SC221-M and the Bacillus cereus BSC24 strain were selected to generate microbial preparations. The results showed that addition of the microbial preparations decreased various hydrochemical parameters, including total dissolved solids, ammonium, nitrite, total nitrogen and the chemical oxygen demand. Nitrogen removal rates were highest on day 9; the removal rates of BSC24, SC221-M, a mixed preparation and a 3× mixed preparation were 24.5%, 26.6%, 53.9% and 53.4%, respectively. The mixed preparation (SC221-M+BSC24) was more effective at removing nitrogen than either the SC221-M or BSC24 preparation. Roche 454 pyrosequencing and subsequent analysis indicated that the control and other groups formed separate clusters, and the microbial community structure in the water changed significantly after the addition of microbial preparations. These results indicate that the addition of microbial preparations can improve both the water quality and microbial community structure in an experimental aquaculture system. P. stutzeri strain SC221-M and its related microbial preparations are potential candidates for the regulation of water quality in commercial aquaculture systems.