In this study, a pure culture of Zoogloea ramigera was grown as a film on active carbon particles in a differential fluidized bed biofilm reactor (DFBBR) as a novel experimental system for the application of diffusion-reaction models. The effective diffusion coefficients of the essential substrates; glucose, ammonium ion and oxygen in the active biofilm were simultaneously calculated. The values of effective diffusion coefficients in the active biofilm to those in water varied between 13-34, 48-96 and 28-48% for glucose, ammonium ion and oxygen, respectively, al different biofilm densities, which is in fair agreement with the literature.