A multiplex lateral flow immunoassay (LFA) is developed for the simultaneous on-site determination of three mycotoxins (aflatoxin B1, zearalenone and ochratoxin A) in corn, rice and peanut. By systematically optimizing the preparation of antibody-gold nanoparticle conjugates, the size of gold nanoparticle and the position of capture antigen, the developed LFA can obtain a visual detection limit of 10μg/kg for aflatoxin B1, 50μg/kg for zearalenone and 15μg/kg for ochratoxin A. For quantitative analysis, the limits of detection were 0.10-0.13μg/kg for aflatoxin B1, 0.42-0.46μg/kg for zearalenone, and 0.19-0.24μg/kg for ochratoxin A, which were far below the regulatory limits set by the European Commission. At the spiked concentrations of 0.5-10.0μg/kg, the mean recoveries of the three mycotoxins ranged from 86.2 to 114.5% with coefficients of variation less than 16.7%. These results demonstrated that the developed immunoassay can be used for routine monitoring of mycotoxin contamination.