Allostery is a fundamental biological phenomenon that occurs when a molecule binds to a protein's allosteric site, triggering conformational changes that regulate the protein's activity. However, allostery in antibodies remains largely unexplored, and only a few reports have focused on allostery from antigen-binding fragments (Fab) to crystallizable fragments (Fc). But this study, using anti-phenobarbital antibodies-which are widely applied for detecting the potential health food adulterant phenobarbital-as a model and employing multiple computational methods, is the first to identify a cyclopeptide (cyclo[Link-M-WFRHY-K]) that induces allostery from Fc to Fab in antibody and elucidates the underlying antibody allostery mechanism. The combination of molecular docking and multiple allosteric site prediction algorithms in these methods identified that the cyclopeptide binds to the interface of heavy chain region-1 (CH) in antibody Fab and heavy chain region-2 (CH) in antibody Fc. Meanwhile, molecular dynamics simulations combined with other analytical methods demonstrated that cyclopeptide induces global conformational shifts in the antibody, which ultimately alter the Fab domain and enhance its antigen-binding activity from Fc to Fab. This result will enable cyclopeptides as a potential Fab-targeted allosteric modulator to provide a new strategy for the regulation of antigen-binding activity and contribute to the construction of novel immunoassays for food safety and other applications using allosteric antibodies as the core technology. Furthermore, graph theory analysis further revealed a common allosteric signaling pathway within the antibody, involving residues Q123, S207, S326, C455, A558, Q778, D838, R975, R1102, P1146, V1200, and K1286, which will be very important for the engineering design of the anti-phenobarbital antibodies and other highly homologous antibodies. Finally, the non-covalent interaction analysis showed that allostery from Fc to Fab primarily involves residue signal transduction driven by hydrogen bonds and hydrophobic interactions.