The layer-by-layer (LbL) assembly as a simple and effective method has been extensively used to prepare polyelectrolyte films but the buildup mechanism is expected to be further clarified. In this work, the structure and formation mechanism of LbL-assembled heparin/chitosan multilayer composite films were characterized by electrochemical system, scanning electron microscope and atom force microscope. The results revealed that the film grew linearly in the first 10 bilayers based on measured linear increase of film resistance with number of layers, while the film grew exponentially in the later 10 bilayers based on measured nonlinear increase of film resistance. The charge-transfer resistance increased in an oscillatory way or a linear way at different growing stages, which was discussed with their formation mechanism and the interfacial structure on electrode. A buildup mode of the LbL film was suggested based on the structural and electrochemical characters.