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CD28 is a costimulatory receptor that provides the second signal necessary for T-cell activation and is associated with diseases, including rheumatoid arthritis, asthma, and cancer. Targeting CD28 is crucial for both functional bioanalysis and therapeutic development. Molecular probes, particularly fluorescent probes, can enhance our understanding of CD28′s cellular roles. However, existing antibody-based probes face challenges such as high production costs, low stability, and large size, which limit their bioanalytical applications. Thus, there is a need for smaller, robust probes that enable the sensitive and selective targeting of CD28. Multicyclic peptides have emerged as promising candidates for novel therapeutics and molecular probes. Recently, we identified disulfide-directed multicyclic peptides (DDMPs) that bind CD28 with submicromolar affinity; however, their relatively low affinity limits further applications. In this study, we develop a DDMP evolving system based on yeast display and error-prone PCR to identify high-affinity peptide binders. We obtained DDMPs with a picomolar affinity for CD28, exceptional binding specificity, and remarkable oxidative folding efficiency. Furthermore, we developed fluorescent probes and labeling strategies for detecting and visualizing CD28 expression in human T cells. This advancement opens new avenues for studying T-cell dynamics and activation states, which are essential for understanding immune responses and developing targeted therapies. Our study not only produces potent CD28 binders and probes but also establishes a robust platform for optimizing other multicyclic peptide-based probes and therapeutics.