A comprehensive investigation was made in this study on the variation of dissolved organic nitrogen (DON) during a whole backwashing cycle of the biofiltration for drinking water treatment. In such a cycle, the normalized DON concentration (C effluent /C influent ) was decreased from 0.98 to 0.90 in the first 1.5h, and then gradually increased to about 1.5 in the following 8h. Finally, it remained stable until the end of this 24-hour cycle. This clearly 3-stage profile of DON could be explained by three aspects as follows: (1) the impact of the backwashing on the biomass and the microbial activity; (2) the release of soluble microbial products (SMPs) during the biofiltration; (3) the competition between heterotrophic bacteria and nitrifying bacteria. All the facts supported that more DON was generated during later part of the backwashing cycle. The significance of the conclusion is that the shorter backwashing intervals between backwashing for the drinking water biofilter should further decrease the DON concentration in effluent of biofilter.