•Label-free aptamer was immobilized on the surface of MNPs via hybridization with capture probe.•A simple and universal aptamer-based fluorescent assay is described for allergen detection.•The magnetic-assisted aptamer-based sensing system showed desired sensitivity and selectivity for tropomyosin detection.•The proposed aptamer-based assay presented a low detection limit of 77 ng mL−1. Herein, a facile and versatile label-free aptamer-based fluorescent assay for simple and sensitive detection of food allergen in food matrix is developed based on the functionalized magnetic nanoparticles (MNPs) as separation carrier and commercially available OliGreen dye as signal probe. First, the aptamer hybridized with capture probe that was conjugated on the surface of MNPs to form aptamer-MNPs complexes as detection probes, which undergo conformational change upon interaction with target analytes, resulting in the release of aptamer from the surface of MNPs. So the released aptamer in the supernatant produced a significant fluorescence enhancement signal by introducing the OliGreen dye based on its ultrasensitive and specific fluorescence enhancement upon binding to ssDNA. As proof-of concept, this proposed strategy was applied to quantitative detection of tropomyosin, one of the major allergen found in shellfish. Under optimal conditions, the linear range was from 0.4 to 5 μg mL−1 (R2 = 0.996) with a low limit of detection calculated to be 77 ng mL−1. Moreover, the aptamer-based assay had remarkable selectivity and was successfully applied in the detection of tropomyosin in food matrix. Due to its simple design, easy operation and high sensitivity and selectivity, the proposed aptamer assay could expect to expand applications in various allergens detection by changing corresponding aptamers on the detection probe.