A multiplex lateral flow immuno-biosensor has been developed for on-site quantification of aflatoxin B1, Fumonisin-B1, and zearalenone in food, necessitating the conjugation of their anti-toxin antibodies to gold nanoparticles (AuNPs). This study aims to optimize physicochemical reaction conditions for stabilizing the conjugates of anti-toxin antibodies with AuNPs. Bio-functionalized AuNPs with anti-toxin antibody conjugates possess unique physical, biochemical, and optical properties that can significantly enhance biochemical assay performance by amplifying signal intensity, improving signal transduction, increasing analyte detection sensitivity, and enabling simple colorimetric signal readout. We prepared an immunochromatographic test strip with lines of distinct colors using AuNPs of different shapes, exhibiting blue, red, and purple hues. A comprehensive synthesis procedure for AuNPs and their conjugates (anti-toxin antibodies) was investigated under varied physicochemical reaction conditions. Three differently sized AuNPs were synthesized to conjugate with mycotoxin antibodies via physisorption and carbodiimide covalent approaches for an AuNPs-based immunochromatographic assay. This study focuses on the conjugation efficiency of aflatoxin-B1, Fumonisin-B1, and zearalenone antibodies with differently sized AuNPs under various reaction conditions. The impact of physicochemical reaction factors such as pH value, antibody concentration, and ratio, and the role of NaCl as a flocculating agent were discussed. Immunogenic haptens of the antigen and their protein conjugates were prepared via the oxime active ester (OAE) method, followed by mycotoxin oxime synthesis through an oximation reaction with CMO. It was observed that differently sized AuNPs exhibited varying conjugation efficiencies under diverse pH conditions and antibody and hapten concentrations. These findings contribute to enhancing on-site mycotoxin detection capabilities.