Abstract: National standard methods were all for the individual determination of a single mycotoxin, rather than the simultaneous determination of multiple mycotoxins. Additionally, the pre-column derivatization required derivatizing reagents; among literature methods, the post-column derivatization also needed additional derivatization devices. For this reason, the method mentioned by the title was proposed. Wheat samples were milled into 425 μm standard test sieve. The mycotoxins were extracted from the powder by using 5 times volumes of 80%（volume fraction） acetonitrile solution, orbital shaking for 30 min or vortex shaking for 20 min. Filtrate was obtained from the mixture by centrifugation and filtration. 5 mL of the above filtrate and 35 mL of phosphate-buffered saline （PBS） solution were mixed and then 3 mycotoxins including deoxynivalenol （DON）, zearalenone （ZEN） and aflatoxin B₁ （AFB₁） were purified simultaneously by using a triple-combined immunoaffinity column instead of a single immunoaffinity column, and a fully automatic solid phase extraction instrument replacing the manual operation during the purification. The purified eluate was concentrated by nitrogen-blowing, and the residue was reconstituted with 30% （volume fraction） methanol solution, and passed through a 0.22 μm filter membrane. 3 mycotoxins in the above filtrate were simultaneously determined by high performance liquid chromatography, using Waters Atlantis^TM T3 C₁₈ column as the stationary phase, and a mixture of methanol and water at different volume ratios as the mobile phase for gradient elution. Among them, DON was detected by the photodiode array detector at the front end, and ZEN and AFB₁ were detected by the fluorescence detector at the back end. While the sample solution in the mobile phase with a high methanol content flowing through the photodiode array detector not only achieved the quantitative detection of DON, but also realized the photochemical derivatization of AFB₁ and the improvement of its fluorescence response value. The method was established for the simultaneous determination of 3 mycotoxins with one-time sample pretreatment and one injection, without additional derivatization devices. Linear relationships between their corresponding peak areas and mass concentrations of 3 mycotoxins were kept in definite ranges, with lower limits of determination of 200 μg·kg⁻¹ for DON, 10 μg·kg⁻¹ for ZEN, and 0.1 μg·kg⁻¹ for AFB₁, respectively, all meeting the requirements specified in the national standards of GB 5009.111—2016 （DON 200 μg·kg⁻¹）, GB 5009.209—2016 （ZEN 17 μg·kg⁻¹）, and GB 5009.22—2016 （AFB₁ 0.1 μg·kg⁻¹）. Test of recovery was made on blank wheat matrices by the standard addition method, giving results in the range of 89.5%−98.8%, with RSDs （n=6） of the determined values less than 7.0%. A t-test of paired-samples was performed on the determination results of 3 mycotoxins, which were obtained by this method and the national standard methods, and it was shown that there was no significant difference between the two methods.