Elemental sulfur autotrophic denitrification (S0AD) processes are temperature-sensitive, presenting a substantial challenge for the practical implementation of S0AD bioreactors. In this study, a comprehensive methodology for designing and operating S0AD bioreactors was developed, effectively managing fluctuations in nitrogen removal efficiency caused by seasonal temperature variations. Initially, the nitrate removal rate was correlated with simulated on-site temperature and nitrate loading, revealing correlation coefficients of k1, k2, k3, and A as 5.42×10−4, −0.41, 0.04, and 0.13, respectively, to establish a mathematical model for predicting S0AD efficiency. Subsequently, by considering influence factors such as dissolved oxygen and dynamic sulfur consumption, the model was employed to accurately design a pilot-scale S0AD bioreactor for a case study. By utilizing an alternative multi-subunit operation, a stable effluent nitrate concentration of less than 8 mg-N/L was maintained throughout the year. Importantly, this approach resulted in a substantial reduction of 76.8% in excessive nitrate removal, sulfur consumption, and sulfate production. This study aims to provide an optimal design and operation strategy for the practical application of S0AD bioreactors, thereby enhancing reliability and cost-effectiveness in the face of seasonal temperature changes.