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In this study, a rapid and sensitive immunoassay method has been established based on calibration curve implanted enzyme-linked immunosorbent assay (C-ELISA) for the simultaneously quantitative determination of aflatoxin B₁, deoxynivalenol and zearalenone in cereal samples, soybean and peanut. The C-ELISA avoids using the standard substances during the detection. The principle of the C-ELISA is to implant the optimized standard curve data into the matched analysis software which can make data processing more convenient and faster. The implanted calibration curve software was programmed with C plus plus. In the new immunoassay system for aflatoxin B₁, deoxynivalenol and zearalenone, their linear detection ranges were from 0.03~0.81, 1.00~27.00 and 5.00~135.00 ng/g, respectively. Recovery rates from spiked samples ranged from 85% to 110% with the intra-assay coefficients of variation under 5%. Compared with HPLC method, the new method showed consistence in all the observed contents of the three mycotoxins in real samples. The new method can rapidly and reliably high throughput simultaneously screen for multiplex mycotoxins.