Aflatoxin B1 (AFB1), one of the most toxic chemical carcinogens, has been widely studied. It remains challenging to develop simple, accurate, and sensitive analytical methods for the detection of AFB1 in food matrixes. In this work, on the basis of a dual-signaling strategy, a ratiometric aptasensor was designed and verified for the accurate and sensitive detection of AFB1. The electrochemical method was first used as a model to verify the specific interaction between AFB1 and the aptamer, in which ferrocene (Fc)-anchored and methylene blue (MB)-anchored DNA sequences acted as dual signals. Consequently, the specific interaction between AFB1 and its aptamer was demonstrated by the "signal-on" mode of Fc and the "signal-off" mode of MB. Due to the simple dual-signaling mode, the electrochemical sensor was further extended to the construction of an electrochemiluminoscence (ECL) aptasensor. In the ECL system, dual ECL signals were produced from CdTe/CdS/ZnS quantum dots (QDs) and luminol. Horseradish peroxidase-modified gold nanorods (HRP/Au NRs) acted as the quencher/enhancer and as such quenched the ECL signal of the QDs by ECL energy transfer and simultaneously catalyzed H2O2 to enhance the ECL of luminol. Owing to the self-calibration by the internal reference, both of the ratiometric aptasensors exhibited accurate and sensitive analytical performance for AFB1 with a good linear range from 5.0 pM to 10 nM and detection limits of 0.43 and 0.12 pM (S/N = 3), respectively. The aptasensors also exhibited good selectivity, reproducibility, and stability, revealing potential applications in food safety monitoring and environmental analysis.