Six dominant microorganisms including four kinds of Pseudomonas and two kinds of Bacillus were isolated from substrate sludge for improving the organic substance removal ability for the biological activated carbon filter (BAC). This paper examines the backwashing effect on the stabilization of these six microorganisms immobilized on the filter. The results showed the backwashing had more impact on bacteria stabilization of BAC filter during start-up period than the stabilization period. The biomass loss was 28.57% during the start-up period more than the stabilization period which biomass loss was 11.76%. The Scan Electron Microscope (SEM) and Denaturing Gradient Gel Electrophoresis of Polymerase Chain Reaction amplified 16S rRNA (PCR-DGGE) photos showed that the ecology of the population did not change with the operation. The biomass of dominant microorganism after air-water scour backwashing decreased during the start-up period and retained stability during long term performance.