[Background] Heterotrophic nitrifying-aerobic denitrifying microorganisms have received more and more attention, because they achieve the vision of simultaneous nitrification and denitrification in one system. The nitrogen removal pathway is different along with different bacteria. While the nitrogen metabolic pathway of bacteria is direct correlation with the type and activity of nitrogen elimination enzyme. So find out the nitrogen removal pathway of Pseudomonas alcaliphila AD-28 will provide technical support in application. [Objective] In order to reveal the nitrogen removal mechanism, the nitrogen removal characteristic and key enzymes for nitrogen degradation of Pseudomonas alcaliphila AD-28 were studied. [Methods] The nitrogen removal characteristic of strain AD-28 was investigated when sodium citrate was used as carbon source, ammonium sulfate, sodium nitrite and potassium nitrate were used as nitrogen sources. At the same time, the activities of key enzymes-ammonia monooxygenase (AMO), hydroxylamine oxidoreductase (HAO), nitrite reductase (NIR) and nitrate reductase (NAR) were measured. [Results] After 24 h treatment by strain AD-28, bacteria density (OD600) reached 1.971, the degradation rates of NH4+-N, NO3?-N, NO2?-N and total nitrogen (TN) were all exceeded 96% when the initial concentrations were 18.85, 26.13, 19.47, 66.11 mg/L, respectively. The corresponding specific activities of AMO, HAO, NIR and NAR were 0.028, 0.003, 0.011, 0.027 U/mg, respectively. [Conclusion] The results suggest that AD-28 is a simultaneous heterotrophic nitrification-aerobic denitrification strain. The nitrogen removal pathway of the stain was deduced as following. NH4+-N was oxidized to NH2OH by AMO, then NH2OH oxidized to NO2?-N by HAO, NO2?-N and NO3?-N removed from the medium by NIR and NAR.