Since aflatoxin B1 (AFB1) is a highly toxic and carcinogenic secondary metabolite, it is of great importance to develop an effective method for detection of it. Based on hybridization chain reaction (HCR), we proposed a ratiometric dual-signal electrochemical aptasensor (DECA) to realize rapid and sensitive detection of AFB1. In the aptasensor, ferrocene (Fc) and methylene blue (MB)-tagged single-stranded DNA sequences acted as dual-signal sources. Based on this, more stable hairpin DNA (HS-H1) and MB-labeled auxiliary hairpin DNA (H1 and H2) were introduced to extend the sensing platform. In presence of AFB1, it will compete with MB-cDNA and specifically combine with Fc-labeled aptamer. As a result, MB-cDNA will release and trigger the HCR for MB signal amplification with an enhancement in sensitivity. Besides, the ratio of current variations behaved a ratiometric signal output mode. In the proof-of-concept study, the single-signal mode behaved a linear range of 1 pM~10 nM with limit of detection (LOD) of 0.56 pM (IMB) and 0.15 pM (IFc), respectively, while the ratiometric detection mode showed the lowest LOD of 0.12 pM (S/N = 3). The good specificity, reproducibility, and stability of DECA further revealed its promising application in food safety monitoring.