Ferrocene (Fc) is a common quencher of Ru(bpy)₃²⁺ luminescence. However, interactions between Fc and Ru(bpy)₃²⁺ can be extremely complicated. In this work, we reported the first use of Fc to regulate the electrochemiluminescence (ECL) of Ru(bpy)₃²⁺ by tuning the length of the DNA sequence between Fc and the luminophore of nitrogen-doped graphene quantum dots-Ru(bpy)₃²⁺-doped silica nanoparticles (SiO₂@Ru-NGQDs). The ECL of SiO₂@Ru-NGQDs was depressed when the distance between Ru(bpy)₃²⁺ and Fc was less than 8 nm; a stronger ECL was observed when the distance was more than 12 nm. The switching of the ECL of Ru(bpy)₃²⁺ by Fc was attributed to the electron transfer mechanism, in which Fc participated in the redox of Ru(bpy)₃²⁺ for "signal-off" ECL; this favored electron transfer at the electrode fabricated with an Fc-labeled aptamer (Fc-apt) and SiO₂@Ru-NGQDs for "signal-on" ECL depending on the length of the DNA sequence. Here, a dual-signal readout aptasensor for aflatoxin B1 (AFB1) detection was developed via the enhanced ECL of SiO₂@Ru-NGQDs by Fc-apt. The redox currents of Fc and the ECL of Ru(bpy)₃²⁺ were simultaneously collected as yardsticks, and both decreased with higher concentrations of AFB1. The aptasensor allowed linear ranges of 3 × 10^-5 to 1 × 10² ng mL^-1 for ECL mode and 1 × 10^-3 to 3 × 10³ ng mL^-1 for electrochemical mode. Our work provides insight into the interactions between Fc and Ru(bpy)₃²⁺. The dual-signal readout strategy is a potential platform for the versatile design of aptasensors.

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