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Signal amplification is one of the most effective ways to develop the high-performance electrochemical sensors. However, it can be more complicated for ratiometric detections. Herein, a ratiometric electrochemical aptasensor for aflatoxin B1 (AFB1) was proposed by taking advantage of a dual-amplification strategy by coupling of DNA walker (DW) with hybridization chain reaction (HCR). The special binding of AFB1 with ferrocene (Fc)-labelled aptamer triggers DW on hairpin DNA (hDNA) tracks to produce abundant double-stranded DNA (dsDNA). HCR-based strand amplification occurs on these dsDNA to absorb more methylene blue (MB). Then current ratio of MB (I MB) and Fc (I Fc) is designed as a yardstick to detect AFB1. Our experiments reveal that the interaction between Fc and MB (i.e., steric hindrance, electron mediator) varies. In addition to steric hindrance, the presence of MB also acts as electron mediator, thereby facilitating the electron transfer between Fc and electrode. Such combined effect consequently depresses the efficiency of dual-amplification strategy to improve the detection. The developed ratiometric electrochemical aptasensor allows the accurate detection of AFB1 in the 0.003–3 pg mL−1 range. Our work has shed light on the amplification strategy for ratiometric sensing, and provided a new route in integrating different amplification strategies. • A dual-amplification strategy was proposed by the coupling of DNA walker and hybridization chain reaction. • The developed aptasensor exhibits superior analytical performance for the detection of aflatoxin B1. • The interaction of probes depresses the efficiency of amplification strategy on the improvement of ratiometric sensing. [ABSTRACT FROM AUTHOR]