The development of a sensory signal amplification approach is very crucial for rapid and precise detection of aflatoxin B1 (AFB1). However, such approaches remain scarce due to the weak interactions between AFB1 and the sensing probes. Herein, the first example of a dual-excitation fluorescent platform for antibody-free AFB1 detection is reported, which is assembled by an ordered π-π stack of cationic perylene derivative (PTHA) and tris(2,2'-bipyridine)ruthenium(II) [Ru(bpy3)2+]. Taking advantage of stepwise assembly and multiple binding sites of the nanoprobe, its ability for capturing AFB1 is significantly improved driven by multiple noncovalent interactions. Interestingly, dual-excitation fluorescent sensing mode with signal superposition and self-calibration is activated in the supramolecular coassembly process. Under excitation of 365 nm and 440 nm, the platform exhibits specific recognition toward AFB1 and the limit of detection is determined to be 0.12 ng mL-1. Notably, the dual-excitation platform demonstrates exceptional sensitivity enhancements of 106-fold, revealing that the self-calibrated reference improves the sensitivity and accuracy of analytical method significantly. The applications of our platform not only crack the problem of precise AFB1 detection via supramolecular coassembly strategy but also provide a universal sensitization strategy for ultrasensitive analysis in complex environments.