Various influences of hydrodynamic parameters and substrate feeding patterns on the biofilm formation in the three-phase fluidized bed bioreactor for the nitrification process were investigated in this study. The results clearly indicated that the biofilm formation was significantly affected by the mean liquid velocity. While the mean liquid velocity increased from 444 to 563 m/h, the biomass attached on to the GAC obviously decreased from 30.4 to 15.6 mgVSS/gGAC due to hydraulic sloughing off. Higher velocity was shown to exert a profound influence on the amount of attached biomass. It was caused by the abrupt intensification of shear stress and frictional forces both acting on the bioparticle surfaces. On the other hand, the nitrifying bacteria were induced to secrete more exopolymers covering the bacterial colony onto the GAC at a higher liquid velocity. It would be useful for accumulation of the low growth-rate nitrifiers to attach better on the biofilm carrier in the bioreactor. Consequently, the suspended biomass was attached onto the GAC again and grown up on the GAC surface after 30 days of operation while the primary biofilm was formed. With long-term operation and microbial acclimation, a high efficiency of ammonium removal was achieved and independent of the volumetric loadings of ammonium in the range of 1.5∼3.8 kgNH4+-N/m3/day. It clearly indicates the potential of the three-phase fluidized bed to treat wastewater with a high volumetric loading of ammonium.