Abstract: Excessive intake of vanillin poses health risks, necessitating the development of accurate detection methods. Highly sensitive and specific monoclonal antibodies are essential for rapid immunoassays. In this study, vanillic acid was employed as a hapten and conjugated with carrier proteins (BSA and OVA) via the active ester method to synthesize vanillin artificial antigens (immunogen and coating antigen). Conjugation was confirmed by ultraviolet full-wavelength scanning and gel electrophoresis. BALB/c mice were immunized with the immunogen, and after four immunizations, the polyclonal antibody titer reached 1:160,000, indicating strong immunogenicity of the artificial antigen. A mouse exhibiting a high titer was selected for cell fusion. Following three rounds of subcloning, two positive hybridoma cell lines (1A2 and 3D4) were obtained. The cells were separately injected into the peritoneal cavities of mice (designated B1 and B2) to produce ascites, from which anti-vanillin monoclonal antibodies were harvested. Indirect ELISA revealed that the titers of both ascites were 1:819,200. Sensitivities were determined by indirect competitive ELISA: the IC50 values for ascites from mouse B1 and mouse B2 were 1610 ng/mL and 290 ng/mL, respectively. Cross-reactivity of the B2 ascites with ethyl vanillin, methyl vanillin, coumarin, BSA, and OVA was below 0.1% in all cases, demonstrating excellent specificity. Collectively, a highly sensitive and specific monoclonal antibody against vanillin was successfully developed, establishing a foundation for vanillin immunoassays.