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Enrichment, separation and purification are very important to accurately analyze mycotoxins in complicated samples. In the work, we developed a new enrichment, purification and high-performance liquid chromatography combined with fluorescence detector (HPLC-FLD) for aflatoxins B₁ (AFB₁), ochratoxin A (OTA) and Zearalenone (ZEN) assay using the macroporous magnetic 3D photonic crystal microspheres (3DPCMs). The conditions of enrichment and purification for mycotoxins have been optimized, which are as follows: pore size of 3DPCMs at 280 nm, 1:1 methanol:acetonitrile (v/v) as eluent, antibody concentrations at 60 µg/mL,60 µg/mL and 120 µg/mL for OTA, AFB₁ and ZEN, respectively. The recovery rates in the rice, wheat and corn samples range from 70.01% to 100.12% and the relative standard deviation (RSD) range from 0.45% to 7.09%. The recovery rates used 3DPCMs are almost tenfold …