Radius filler is a kind of filling material used in triangle gap of composite beam and rib structure. To optimize the hand-made preparation method of radius fillers and provide the basis for engineering practice and automatic production, the prepreg radius fillers were prepared by rolled mode and laminated mode respectively. To investigate the influence of different hand-made preparation methods on quality of radius fillers, the boundary dimension, microstructure and mechanical property of prepreg radius fillers were characterized. The results show that there is little difference in boundary dimension of the prepreg radius fillers prepared by rolled mode and laminated mode. The fiber volume fraction is all around 60%, and the void content is all around 3%. But the form of inner defects and mechanical property of the radius fillers differ significantly. For the radius fillers obtained by rolled mode, the inner defects are small and scattered, fibers arrange in disorder and bending failure mode is fiber fracture. For the radius fillers obtained by laminated mode, the inner defects are large and continuous, fibers arrange neatly and collimated, the bending failure mode is delamination fracture.