Background: SDF-1/CXCR4 signaling plays key roles in directed cell migration under physiological and pathological conditions. To develop agonist-based CXCR4 probes for detection of CXCR4 expression on cell lines and metastatic tumors, SAR analyses of fluorescent SDF-1 derivatives were carried out. Results: Several SDF-1 derivatives with a single fluorescent label were designed and synthesized. Modification of the SDF-1 C-terminus with AlexaFluor^® 488 or tetramethylrhodamine provided potent CXCR4 probes. Using a potent probe, a novel binding inhibition assay was established for biological evaluation of potential CXCR4 ligands. Conclusion: SDF-1 derivatives with C-terminal modification exhibit equipotent binding with CXCR4 and an alternative SDF-1 receptor CXCR7 to unlabeled SDF-1. The SDF-1 derivatives are applicable to flow cytometry to detect the receptor expression and identify binding compounds for CXCR4.