Abstract: To improve the adhesion performance of nitrifying bacteria and denitrifying bacteria on zeolites surface and achieve the purpose of efficient removal of ammonium in initial roof rainwater and prolonging the service life of bio-zeolites, experiments of strengthening bio-zeolites formation by acyl-homoserine lactone (AHLs) were carried out. Results show that the adhesion abilities of nitrifying bacteria WGX5 and WGX13 are improved obviously when 2.0 μmol/L C12-HSL is added (The attachment biomass of WGX5 and WGX13 increases by 89% and 94%, respectively). However, the adhesion abilities of denitrifying bacteria HF2 and HF8 were not improved obviously compared with nitrifying bacteria. When the ratio of carbon to nitrogen is 4, and 2.0 μmol/L C12-HSL was added, the adhesion abilities of denitrifying bacteria HF2 and HF8 were improved obviously, and the attachment biomass of HF2 and HF8 increases by 1.3 times and 1.6 times, respectively. The bio-zeolites prepared under the conditions can effectively remove the ammonium in the initial roof rainwater in a long term (the removal efficiency of ammonium in 5 rounds of experiments is as high as 91%-98%). Moreover, the chemical oxygen demand (COD) in the initial roof rainwater is removed partially (the removal efficiency of COD in 5 rounds of experiments reaches 61%-70%). In addition, the in-situ regeneration efficiency of the bio-zeolite enhanced biofilm formation increases from 34% to 83%, thus, the service life of the zeolite is prolonged effectively. In the 5 rounds of experiments, the bio-zeolite enhanced biofilm formation can effectively removed the ammonium from the roof rainwater.