Currently most wastewater treatment plants are faced with upgrading and reconstruction to further improve the effect of deep denitrification. To this end, this paper conducts an experimental study on deep denitrification in the sulfur mixotrophic nitrification filter process using sulfur as filler. During the experiments, when the water temperature of the mixed pool was around 15℃, the influent nitrogen load was 7.3×10-3kg/m3• d, and hydraulic retention time (HRT) was 3.5 h, the average effluent TN concentration was 4.22 mg/L and the TN removal rate reached 43.42%, stably reaching the requirements of the core control area stipulated in the Daqing River Basin Water Pollutants Discharge Standard (DB13/2795-2018); under the conditions of HRT = 3.5 h and water temperatures of 15℃, 22℃, 27℃, 35℃ and 40℃, the average TN removal rate of the mixotrophic denitrification filter reached the highest at 35℃, up to 63%; two-factor analysis of variance (ANOVA) was applied to illustrate the influence of the two factors (water temperature and HRT) on the TN removal rate, and to conclude that the influence of each factor on its TN removal rate is ranked in a descending order: B (water temperature) > A (HRT) > A × B (interaction), all of which were important conditions to be considered, and water temperature was the most influential. These results show that the sulfur mixotrophic denitrification filter has the ability to deeply degrade the TN in the secondary effluent. This study provides a theoretical basis for the sulfur mixotrophic denitrification filter to be used as a deep treatment unit. Also, in the actual application, other operating conditions should be designed and considered to adapt to the uncertain seasonal temperature changes.