Mycotoxins are detectable in 60-80% of food crops, posing significant threats to human health and food security, and causing substantial economic losses. Most mitigation approaches focus on detecting mycotoxins with standard methods based on liquid chromatography coupled with mass spectrometry (LC-MS). Typical MS methods require extensive sample preparation and clean-up due to the matrix effect, followed by time-consuming LC separation, complicating the analysis process and limiting analytical throughput. This study reports the development of a repackable microfluidic molecularly imprinted solid-phase extraction coupled with mass spectrometry (μMISPE-MS) method for rapid detection of zearalenone in agri-food samples. Silica microspheres coated with molecularly imprinted polymers were synthesized as the sorbent for analyte enrichment and sample clean-up. A cost-effective microfluidic chip was designed and fabricated as the μMISPE platform with fully automated operation, including on-line microcolumn packing and unpacking. With optimized solvent conditions and on-chip μMISPE protocol, the entire analytical process from sample to answer was completed within 15 min and achieved high recoveries (71-94%) for corn and rice samples at residue levels of 0.05-0.5 ppm (within Canadian regulatory limits of 0.2-10 ppm). This μMISPE-MS method provides a promising tool for improving mycotoxin monitoring in agri-food systems and is generalizable to other rapid analyses of targeted chemicals in complex matrices.