There has been increasing recent concern about the agricultural use of organophosphorus pesticides. A rapid and sensitive fluorescence assay for the detection of three organophosphorus pesticides has therefore been developed using 6carboxyfluorescein labeling aptamer as the probe and functionalized magnetic nanoparticles as the separation carrier. The aptamer hybridized with complementary DNA conjugated on the surface of the magnetic nanoparticles to form a magnetic aptamercomplementary DNA complex. Upon introducing the target organophosphorus pesticide, the aptamer departed from the complementary DNA, resulting in the fluorescence signal. Under optimized conditions, the limits of detection (LODs, SN 3) for trichlorfon, glyphosate, and malathion were 72.20 ng L1, 88.80 ng L1, and 195.37 ng L1, respectively. The method was applied for the detection of trichlorfon, glyphosate, and malathion in spiked lettuce and carrot samples. The recoveries were in the range of 79.4%118.7%, which were in good agreement with those obtained by gas chromatography, and the relative standard deviations were also acceptable. The method therefore has high sensitivity, so provides a means for the detection of multiple organophosphorus pesticides.