In this study, ozone treatment was applied to modify poly(L-lactic acid) (PLLA) with the intermediate reagent acryl-N-succinimide (ASI). Then, P15, the peptide related to the attachment and differentiation of osteoblastic cells, was reacted with ASI. Ozone activation successfully created peroxides on the surface of PLLA, which was quantitatively determined by the iodide method. By changing the activation temperature, oxygen flow rate, reaction bath, reaction temperature or addition of ferrous ions, the amount of peroxides was controlled and the effects of these variables were explored in this research. The immobilizations of ASI and P15 were confirmed and quantitative analyzed by FT-IR spectroscopy, elementary analysis and amino-acid analysis. Also, the optimization for ozone activation and ASI grafting were performed. From in vitro experiments, the cultured ROS cells expressed significantly higher ALPase activity and calcium deposition after P15 immobilization. The results demonstrated that the ROS cells expressed osteoblastic phenotypes more significantly when cultured with the substrate modified with P15. In this study, PLLA was successfully modified with P15 by ozone activation and the modification promoted the osteoconductivity of PLLA substrates, which could be helpful in bone tissue engineering.