<p>Aptamer-modified magnetic nanoparticles (Apt-MNPs) were synthetized and used as a magnetic solid-phase extraction (MSPE) sorbent for the selective isolation of α-lactalbumin (α-LA), an allergenic protein from cow’s milk. For this purpose, a thiol-terminated Apt was immobilized onto the surface of vinyl-functionalized MNPs via "thiol-ene" click chemistry reaction. The resulting Apt-modified MNPs were characterized and several parameters that affect the Apt-based MSPE efficiency were investigated. Under the optimal conditions, the developed Apt-based MSPE protocol was applied to the determination of α-LA in different foodstuffs (dairy-free products and those with declared milk content), yielding satisfactory recoveries (80.0–96.9%), good precision (RSD &lt; 5.5%), and a low limit of detection (0.04 μg·mL⁻¹). Moreover, the Apt-based sorbent proved to be highly affordable, with a cost of less than 4.2 € per sorbent, further supporting its suitability for routine analysis.</p> Graphical Abstract <p></p>