Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagent immobilization, prolonged incubations, and product separation. We proposed an approach for aptamer affinity determination by FA taking into account the difference in label fluorescence before and after complexation. The detailed step by step scheme using a native and fluorescently labeled ligand was described and justified in the paper. The scheme ensures the exclusion of data with low reliability and establishes valid criteria for selecting optimal concentrations of reagents (labeled ligand and aptamer) used in the experiments. The approach was experimentally tested using ochratoxin A (OTA), its fluorescein-labeled derivative (OTA-Flu), and the aptamer binding them. We demonstrated that it allows minimizing the influence of fluorescence change to accurately determine the dissociation constant. On the basis of FA registration, the binding constants of the aptamer-OTA-Flu and the aptamer-OTA complexes were found to be equal to 245 + 33 and 63 + 18 nM, respectively. The value for the aptamer-OTA complexes was confirmed by the equilibrium dialysis technique. The resulting constant was 80 ± 9 nM. The versatility and methodological simplicity of the proposed protocol, as well as the short implementation time, are why it can be recommended as an effective tool for characterizing aptamer-ligand complexes.