Molecular imaging was developed from basic molecular recognition. It can visualize not only the expression levels of specific molecules in a living system but also specific biological processes, thus providing guidance for early detection and treatment of diseases. As a noninvasive method, imaging agents are one of the foundations of high spatial resolution imaging, and their sensitivity and specificity can be improved by coupling targeting ligands to imaging probes. Among the various targeting ligands (antibodies, aptamers, etc.), targeting peptides are widely used in various modalities of molecular imaging due to their high affinities toward the molecular target and their excellent physicochemical properties. In this review, we summarize the design concepts and methods of targeting peptides in molecular imaging, introduce the combination of targeting peptides and imaging probes in different imaging modalities (e.g., fluorescence imaging, radionuclide imaging), and provide examples of their applications in bioimaging. Finally, the challenges and strategies for clinical translation and practical application of targeting peptide-based imaging reagents are briefly discussed.