Nitrogen pollution of surface water is the main cause of water eutrophication, and is considered a worldwide challenge in surface water treatment. Currently, the total nitrogen (TN) content in the effluent of wastewater treatment plants (WWTPs) is still high at low winter temperatures, mainly as a result of the incomplete removal of nitrate (NO₃⁻-N). In this research, a novel aerobic denitrifier identified as Pseudomonas sp. 41 was isolated from municipal activated sludge; this strain could rapidly degrade a high concentration of NO₃⁻-N at low temperature. Strain 41 completely converted 100 mg/L NO₃⁻-N in 48 h at 15 °C, and the maximum removal rate reached 4.0 mg/L/h. The functional genes napA, nirS, norB and nosZ were successfully amplified, which provided a theoretical support for the aerobic denitrification capacity of strain 41. In particular, the results of denitrification experiments showed that strain 41 could perform aerobic denitrification under the catalysis of NAP. Nitrogen balance analysis revealed that strain 41 degraded NO₃⁻-N mainly through assimilation (52.35%) and aerobic denitrification (44.02%), and combined with the gene amplification results, the nitrate metabolism pathway of strain 41 was proposed. Single-factor experiments confirmed that strain 41 possessed the best nitrogen removal performance under the conditions of sodium citrate as carbon source, C/N ratio 10, pH 8, temperature 15–30 °C and rotation speed 120 rpm. Meanwhile, the bioaugmentation test manifested that the immobilized strain 41 remarkably improved the denitrification efficiency and shortened the reaction time in the treatment of synthetic wastewater.