Abstract <p>In this study, the interaction of an aptamer having a loop structure (synthetic receptor based on single-stranded DNA) with a specific ligand aflatoxin B1 (<b>AFB1</b>), which is a common toxic contaminant of food products, was studied. The effect of mono- and bivalent cations (Cs⁺, Li⁺, Na⁺, K⁺, Sr²⁺, Ba²⁺, Mg²⁺, Ca²⁺) at different concentrations on the reactivity of the aptamer was studied. The fluorescence anisotropy (<b>FA</b>) of the fluorescein-labeled AFB1, reflecting both binding to the aptamer and the mobility of the aptamer-labeled AFB1 complex in this reaction medium, was recorded. It was shown that the FA changes differed significantly depending on the cation in the reaction medium and its concentration. The effect of monovalent cations on FA was observed in the range of 400 mM–2.5 M. For divalent cations, the influence was more pronounced in amplitude and corresponded to concentrations from 6 to 200 mM. According to the extent of FA changes, cations form the following series: Cs⁺ <InlineEquation ID="IEq1"> <EquationSource Format="TEX">\( \ll \)</EquationSource> <!--AppBio2560171Samokhvalov-m1--> </InlineEquation> K⁺ ≤ Li+ &lt; Na⁺ and Ba²⁺ &lt; Sr²⁺ &lt; Ca²⁺ &lt; Mg²⁺. Comparison of the characteristics of AFB1 competitive determination for the traditionally used reaction medium and the one with a 50-fold increase in the concentration of magnesium acetate (from 20 mM to 1.0 M) showed a 12-fold decrease in the detection limit (down to 2.5 ± 0.4 nM). The obtained results allow considering the cation selection as an effective tool in the development of highly sensitive aptamer-based analytical systems.</p>