[Objective] A combination of Plackett-Burman Design (PBD) with Uniform Design (UD) was applied to optimize the nitrogen removal performance of immobilizing Micrococcus roseus. [Methods] Polyvinyl alcohol and Sodium alginate were used to immobilize cells. Using PBD, five factors including PVA, SA, temperature, the inoculation amount and the amount of pellet were selected from nine factors based on their significance (P<0.05). Next, UD was used to further optimize these five significant factors in order to improve nitrogen removal effect. Further, these data were analyzed by stepwise regression and second-order polynomial, which were based on Minitab and Matlab. [Results] The optimum conditions are as follows: PVA 9.6%, SA 2.2%, 33.4 °C, the inoculation amount 8%, the pellet number 500 per 100 mL, and the highest value of nitrogen removal rate peaked 60.9% after 72 h operation for nitrogen removal. [Conclusion] The combination of PBD with UD is an effective way to select significant factors for the optimization of denitrification performance of Micrococcus roseus. And Micrococcus roseus removes ammonia mainly through assimilation, nitrification and denitrification.