Fluorescent contrast agents are instrumental in amplifying signals, thereby enhancing the sensitivity and accuracy of live optical imaging. However, a significant proportion of traditional fluorescent contrast agents exhibit drawbacks such as short half-life, suboptimal biocompatibility, and inadequate tumor targeting, all of which impede effective imaging guidance. Peptides, derived from natural structures, offer a flexible modular design that can be precisely engineered and adjusted using synthetic methods to achieve specific biological activity and pharmacokinetic properties. They bind with designated receptors to exert their effects, demonstrating high specificity. The development of fluorescent probes based on peptides significantly overcomes the limitations of conventional contrast agents, offering superior performance. This article provides a comprehensive review of three strategies for constructing peptide-based fluorescent probes, delving into their distinct design concepts, mechanisms of action, and innovative aspects. It also highlights the potential applications of peptide-based fluorescent probes in tumor diagnosis and image-guided surgery, offering insights into their future clinical transformation.