In this study, the impact of different aeration sequences, hydraulic retention time (HRT), aeration time ratios and external carbon types on performance of biofilters treating secondary effluent during start-up was explored. For an intermittently aerated nitrifying filter, the adjustment of aeration sequence with prior nitrification or denitrification had no significant impact on overall performance. Extending HRT promoted sedimentation and denitrification. However, extending HRT to enhance performance is inadvisable due to incompleted denitrification. Similarly, the impact of different aeration time ratios on performance was not as apparent as that of start-up time. External carbon addition to denitrifying filter could enhance performance with glucose more favorable for denitrification and sodium acetate better for P sequestration. When the synthetic over-standard secondary effluent treated by the nitrifying and denitrifying filters in order with the latter added with glucose, all the monitored indices could reach the A standard (GB 18918-2002, China).