Oral collagen peptide could raise the density of the fibroblasts and increase the diameter and density of collagen fiber in derma of the skin to improve the skin quality. If the collagen peptide was directly applied to the skin, it must passe through the skin to activate the fibroblasts of dermis and to promote the collagens synthesis. In this study, the bioactivities of collagen peptides including activation ability of the fibroblasts and permeation ability of the skin were investingated. Collagen peptides were extracted and protease hydrolyzed from fish scales. The proliferation activities of the skin fibroblast induced by collagen peptides were evaluated by MTT assay. When the concentration of collagen peptides was 200 µg/mL, the proliferation activities of fibroblasts could be enhanced to 150% and the collagen synthesis ability could be increased to 250%. Four types of molecular weight (Mw) 4500, 3500, 2000 and 1300 Da were used for the investigating of the transdermal permeation ability through the Franz type diffusion cell model. The results showed that all sizes of the peptide samples could permeate into dermis. However, those at average Mw 3500 Da and 4500 Da collagen peptides had the higher amount of transdermal permeation than the others. In order to study the permeation of collagen peptides in the nude mice skin, the collagen peptides were labeled with fluorescence dye and then observed by confocal scanning laser microscopy. The fluorescence images showed that the collagen peptides ranging with Mw 3500 Da and 4500 Da had longer, more folding and coiling shapes, while those at average Mw 2000 Da and 1300 Da showed the shorter, less folding and straight shapes. These results suggested that application of the fish scales collagen peptides on skin could also activate fibroblasts and accelerate the collagen synthesis in the dermis to improve skin quality.