We describe a simple aptamer assay for the rapid analysis of aflatoxin B1 (AFB1), a potently carcinogenic fungal metabolite. The original anti-AFB1 aptamer was rationally truncated, obtaining a short aptamer that displayed better performance. Different concentrations of AFB1 were detected by measuring the circular dichroism spectra of this short aptamer. The detection limit reached 0.6 nM, which was lower than that of the original aptamer (80 nM).