Integrin α5β1 is a widely-recognized target for molecular probes in various pathological conditions, especially cancer. The development of computer screening approaches to identify novel high affinity ligands to tumor markers has paved the way for a new generation of tumor identification technology. In this study, we have developed an efficient pharmacophore-based computational strategy to screen two novel peptides, RYr and H5, with high affinity to integrin α5β1. Noninvasive optical imaging data showed that these two peptides could be specifically uptaken by α5β1 overexpressed-tumor cells in vitro and in vivo. And these peptides-based probes could retain in tumor tissue for precise tumor identification. Results indicated that the newly identified peptides with high affinity to integrin α5β1 can be used for precise tumor identification and treatment. And this work exploited more functionalities of pharmacophore-based computational strategy for screening targeting-peptides.