Abstract: The bacteria Rhodococcus ruber is usually used in waste water and sewage treatment. In order to discuss the effect of environmental factors on R. ruber HDRR2Y's removing ammonia nitrogen and nitrite, we added the culture medium of R. ruber strain HDRR2Y to the aquaculture water containing high concentration of ammonia nitrogen (NH₄ ⁺-N) and nitrite (NO₂ ⁻-N), and observed the changes of ammonia nitrogen and nitrite concentrations by the national standard method, so as to investigate the effect of the culture medium on the removal of ammonia nitrogen and nitrite. In addition, we carried out the Plackett-Burman experimental design according to five factors: temperature, rotating speed, salinity, inoculum amount, substrate (Ammonia nitrogen and nitrite) concentration, to explore the effect of these factors on the removal of ammonia nitrogen and nitrite by R. ruber. The results show that during the fermentation process, the mass concentration of strain HDRR2Y increased from 5×10⁴ CFU·mL⁻¹ to 4.08×10⁹ CFU·mL⁻¹ in the initial 36 h. After adding the culture medium, the mass concentration of ammonia nitrogen decreased from 15 mg·L⁻¹ to 5.56 mg·L⁻¹, with a removal rate of 62.96%, and that of nitrite decreased from 15 mg·L⁻¹ to 6.95 mg·L⁻¹, with a removal rate of 59.37%. Among the five factors, temperature and ammonia nitrogen concentration had the most significant effects on the removal of ammonia nitrogen by strain HDRR2Y (P<0.05) (Temperature>ammonia nitrogen concentration>rotation speed>biomass>salinity). Temperature and rotating speed were the two most significant factors affecting the removal of nitrite (P<0.05) (Temperature>rotating speed>salinity>nitrite concentration>bacterial count). It is showed that the culture medium of strain HDRR2Y is good for removing ammonia nitrogen and nitrite, and temperature is the most significant factor affecting the efficiency of HDRR2Y in removing ammonia nitrogen and nitrite.