Abstract: Excessive intake of vanillin causes potential health risks, thereby driving the demand for accurate and efficient detection methods. High-sensitivity and high-specificity monoclonal antibodies are core reagents for the rapid immunoassay of food additives. In this study, vanillic acid was used as a hapten and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) via the active ester method to synthesize artificial vanillin antigens (immunogen and coating antigen). The successful conjugation of hapten and carrier proteins was verified through ultraviolet full-wavelength scanning and gel electrophoresis. BALB/c mice were immunized with the prepared immunogen. After four rounds of immunizations, the titer of the induced polyclonal antibody reached 1∶160 000, confirming the excellent immunogenicity of the synthetic artificial antigen. The mouse with the highest antibody titer was selected for cell fusion. After three rounds of limiting dilution subcloning, two stable positive hybridoma cell lines (1A2 and 3D4) were successfully screened. The two cell lines were separately injected into the peritoneal cavity of healthy mice (designated B1 and B2) to induce ascites production, and anti-vanillin monoclonal antibodies were subsequently purified from the ascitic fluid. Indirect ELISA results showed that the antibody titers of both ascites samples reached 1∶819 200. The sensitivity of the prepared monoclonal antibodies was evaluated by indirect competitive ELISA. The IC₅₀ values of antibodies derived from the two cell lines were 1 610 ng/mL and 290 ng/mL, respectively. Additionally, the ascites of the 3D4 cell line (B2) exhibited negligible cross-reactivity (below 0.1%) with ethyl vanillin, methyl vanillin, coumarin, BSA, and OVA, demonstrating outstanding specificity. In conclusion, highly sensitive and specific anti-vanillin monoclonal antibodies were successfully prepared in this work, this work provides a solid foundation for the establishment of rapid immunoassay methods for vanillin detection.