In this paper, a magnetic separation assisted fluorescence method for the determination of three common organophosphorus pesticides (OPPs) in ‘Shatangju’ mandarin was established. Magnetic beads were synthesized by solvothermal method and modified by polypyrrole (PPy) to form Fe₃O₄@PPy nanoparticles as a fluorescence quenching agent. This method was based on the use of a 6-carboxyfluorescein (FAM)-labeled aptamer (S4-29) by virtue of its broad-spectrum recognition ability for OPPs. When Fe₃O₄/PPy nanoparticles mass ratio, Fe₃O₄@PPy nanoparticles concentration and incubation time were 1:1.5, 0.2 mg/mL, and 120 min, respectivley, the detection limits of phorate, omethoate and profenofos were 4.17 × 10^-4, 2.77 × 10^-4, and 4.67 × 10^-4 μg/kg, and the recoveries ranged from 79.94% to 108.00% with relative standard deviations (RSDs) between 3.05% and 7.39%. In conclusion, this method has strong selectivity and high accuracy and can be used for the analysis of banned OPPs in agricultural products.