Abstract: In order to explore the molecular interaction between recombinant protein cohesin module and dockerin module and its effect on the assembly efficiency. In this study,scaffoldins and enzymes was secreted by Saccharomyces cerevisiae cells,and the interaction of secondary scaffolins and cellulases combined with primary scaffolin was determined and analyzed by non-denatured electrophoresis and isothermal titration calorimetry in vitro. The results showed that the affinity force between the dockerin module of secondary scaffold protein and cohesion module of cellulase protein decreased while the affinity constant（Kd） increased. The assembly reaction was driven by enthalpy change and hydrogen bonds are generated. The results proved that the decrease of intermolecular affinity of the proteins was the main factor that leads to the lower assembly efficiency of secondary scaffold proteins and primary scaffold proteins.