Sulfur-based autotrophic denitrification is a novel biological denitrification process characterized by the absence of an organic carbon source, a short reaction time, a high denitrification rate, a low treatment cost, and a small footprint. However, the technique is facing challenges with respect to engineering applications. In this study, a pilot-scale sulfur-based autotrophic denitrification system was established with an optimal hydraulic retention time (HRT) of 0.21 h, which achieved the highest denitrification load of 1158 mg/(L·d) and a denitrification rate of 164 gNO3−-N/(m3·h). Effective backwashing is the basis for the long-term stable and efficient nitrogen removal performance, which recovered its normal nitrogen removal performance within 0.5 h. In addition, the operation cost is merely 0.013 $/t, indicating that the sulfur-based autotrophic denitrification process presents good economic applicability, and the relatively low operation cost will lay a foundation for practical application.