Aflatoxin B1 (AFB1) is one of the most contaminated fungal toxins worldwide and is prone to cause serious economic losses, food insecurity, and health hazards to humans. The rapid, on-site, and economical method for AFB1 detection is need of the day. In this study, an AFB1 aptamer (AFB1-Apt) sensing platform was established for the detection of AFB1. Fluorescent moiety (FAM)-modified aptamers were used for fluorescence response and quenching, based on the adsorption quenching function of single-walled carbon nanohorns (SWCNHs). Basically, in our constructed sensing platform, the AFB1 specifically binds to AFB1-Apt, making a stable complex. This complex with fluorophore resists to be adsorbed by SWCNHs, thus prevent SWCNHs from quenching of fluorscence, resulting in a fluorescence response. This designed sensing strategy was highly selective with a good linear response in the range of 10–100 ng/mL and a low detection limit of 4.1 ng/mL. The practicality of this sensing strategy was verified by using successful spiking experiments on real samples of soybean oil and comparison with the enzyme-linked immunosorbent assay (ELISA) method.