Nitrogen removal under low C/N ratio in cold season is an arduous task. In this study, riboflavin was used as an eco-friendly electron mediator to improve the denitrification process in an SBR reactor under conditions of low temperature (10 °C to 15 °C) and limited carbon source (C/N ratio = 4.0). The results indicated that riboflavin created a suitable pH in the system for denitrification. Under water temperature of 10 °C to 15 °C, riboflavin (10 mg/L) stimulated the NO3–N and TN removal rate by 16.5%, and 51%, respectively. Riboflavin promoted the utilization efficiency of limited carbon source, driving the denitrification process with low residual acetate as electron donor. The rising cost of riboflavin supplement (10 mg/L) was 0.025 USD per m3 of wastewater. To satisfy the efficient nitrogen removal from municipal wastewater, the optimum C/N ratio and the selection of solid/immobilized redox mediators should be developed in future work.