Sulfur autotrophic denitrification (SAD) using elemental sulfur as the electron donor has aroused increasing interest of its application in treating secondary effluent from wastewater treatment plants (WWTPs). However, high influent dissolved oxygen (DO) in secondary effluent would limit the SAD process. This study examined the effect of different DO concentrations on SAD. Results revealed that both low (0-0.5 mg/L) and moderate (2.5-3.5 mg/L) DO concentrations would not harm the nitrate removal rate (NRR) (p > 0.05). However, high DO concentration (5.5-6.5 mg/L) significantly decreased the NRR (p < 0.05) through strong competition over the nitrate for electrons and cutting the relative abundance of sulfur-oxidizing bacteria (SOB). Both modeling and experimental results showed that applying internal reflux could serve as a strategy to mitigate the negative effect of high DO concentration, while keeping an appropriate ratio was crucial. When treating real membrane bioreactor (MBR) effluent with high DO concentration (5.5-6.5 mg/L), an internal reflux ratio of 0.5 boosted the NRR by 1.5 times. This study provided potential reference and strategy for dealing with high DO concentration wastewater by applying SAD technology.