G protein-coupled receptors (GPCRs) play important roles by transmitting signals when they bind to specific ligands in human. Dysregulation of the GPCRs has been associated to metabolic diseases, inflammatory and cancers, and making them key targets for therapeutic intervention. The structural characterization of GPCR-ligand interactions remains challenging due to the difficulty in obtaining complex structures. In this study, we chose CXC chemokine receptor 4 (CXCR4), a member of the GPCR family, as the receptor and employed AlphaFold3 to predict the interaction sites between ligands and GPCRs. The results show that the extracellular loop 2 (ECL2) region is crucial for CXCL12-CXCR4 interactions. Using this epitope-guided approach, we selected antibodies from a combinatorial library that bind to CXCR4 and block CXCL12 signaling. Two antibodies, C5 and F4, were found to inhibit CXCL12 signaling in reporter cell lines. Furthermore, these antibodies also exhibited antibody-dependent cellular cytotoxicity against the acute T cell leukemia cell line and the B cell lymphoma cell line. This approach provides a promising way to develop effective antibodies for treating CXCR4-expressed cancer cells, as well as for other diseases linked to GPCR dysfunction.