An aptamer-functionalized stir bar sorptive extraction (SBSE) coating is described for the first time devoted to selective isolation and preconcentration of an allergenic food protein, concavanalin A (Con A), followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) determination. For this purpose, the polytetrafluoroethylene surface of commercial magnetic stir bars was properly modified and vinylized to immobilize a thiol-modified aptamer against Con A via straightforward “thiol-ene” click chemistry. The aptamer-functionalized stir bar was employed as SBSE sorbent to isolate Con A, and several parameters that can affect the extraction efficiency were investigated. Under the optimized conditions, Con A was extracted and desorbed during 30 and 45 min, respectively, at 25 °C and 600 rpm. The SBSE MALDI-TOF-MS method provided limits of detection of 0.5 μg mL−1for Con A. Furthermore, the SBSE coating was highly selective to Con A compared to other lectins. The developed method was successfully applied to the determination of low levels of Con A in several food matrices (i.e., white beans as well as chickpea, lentils, and wheat flours). Recoveries ranged from 81 to 97% with relative standard deviations below 7%. The aptamer-based stir bars presented suitable physical and chemical long-term stability (1 month) and a reusability of 10 and 5 extraction cycles with standards and food extracts, respectively. The developed aptamer-affinity extraction devices open up the possibility of developing novel highly selective SBSE coatings for the extraction of proteins and peptides from complex samples.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s00604-023-05795-y.