The research investigated nitrate removal from micro-polluted surface water by the single-stage process of anoxic biofilter using light-weight polystyrene beads as filtration media. In this study, sodium acetate was used as an external carbon source and the nitrate removal efficiency under different regimes of hydraulic loading rate (HLR), water temperature, and C/N ratio was studied. In addition, the effect of backwash on denitrification efficiency was investigated. The results show that the biofilter achieved a high nitrate removal efficiency in 2 weeks at water temperatures ranging between 22 and 25 °C at a C/N ratio (COD:NO3−-N) of 6:1. Besides, the average removal efficiency of nitrate at HLRs of 5.66, 7.07 and 8.49 m3 m−2 h−1 were 87.5, 87.3 and 87.1%, respectively. The average removal efficiency of nitrate nitrogen was 13.9% at a HLR of 5.66 m3 m−2 h−1 at water temperatures of 12–14 °C, then it increased to 93.7% when the C/N ratio increased to 10. It suggests that the optimal hydraulic retention time is at water temperatures of 8–10 °C. The water consumption rate of backwash was about 0.2–0.3%, and denitrification efficiency returned to the normal level in 12 h after backwash.