Contamination of foods by aflatoxin B1 (AFB1) is a common serious problem. To improve the efficiency of AFB1 detection, this study aims to develop a sensitive aptasensor for detection of AFB1 in food samples based on the catalytic effect of aptamer/G-quadruplex DNAzyme probe. The resulting reassembly of this probe in the presence of hemin and K+ catalyzes the generation of fluorescent 2,3-diaminophenazine (DAP) from o-phenylenediamine (OPD). Interestingly, we first found that the high background induced by the superfluous hemin can be effectively suppressed with the aid of sequential adsorption and magnetic separation by magnetic oxidized multiwall carbon nanotubes (Fe3O4@oMWCNTs). This aptasensor exhibits a high sensitivity toward AFB1 with a detection limit of 0.02 ng/mL. The assay also shows higher selectivity for AFB1 compared to other reported agents and can be employed to detect AFB1 in foodstuffs, which might find broad practical applications in other food contaminants determination.