Stormwater runoff greatly contributes to surface water pollution and deterioration of aquatic environment in urban areas. Biofilter, as low impact development (LID) technology, has been widely applied in controlling urban runoff pollution in recent years. A multi-layered biofilter with a saturated zone was employed in this study to test its removal efficiency of pollutants and the effect of saturation time on its performance with the real road runoff. Two groups of columns were built with different vegetations (Zoysia matrella, ZM and a combination of Iris pseudacorus and Z matrella, IZ). During a 2.5-month duration, five times of runoff dosing trials with collected stormwater were conducted at a load of 20-25 L day(-1). All filter systems had effective retention of particulate matters, with the turbidity removals of over 95%. The IZ group performed more efficiently than ZM systems in the removal of total phosphorus (TP), ammonia (NH4-N), nitrate (NO3-N) and turbidity. The mean removal of total nitrogen (TN) and TP was 49.8 +/- 23.8% and 74.8 +/- 9.9% in IZ group, and 42.6 +/- 22.0% and 66.4 +/- 12.2% in ZM group. Saturation time had significant effect on the removal of TN, TP and turbidity, whose concentrations declined with the saturation duration. The performances of biofilters were dynamic, as a function of media depth and retention time. The configuration of saturated zone and optimized depth of media layers (at least 0.75 m) can provide a favorable condition for nitrogen removal under random inflow condition. (C) 2017 Elsevier B.V. All rights reserved.