In this study, ultraviolet (UV) spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking were utilized to systematically elucidate the binding mechanism between aptamers and ochratoxin A (OTA) at different solution conditions (pH, metal ions, and Ca²⁺ concentrations). The results indicated that the optimal pH for aptamer binding to OTA was 6–7. In the presence of Ca²⁺, the primary binding mode between aptamers and OTA was intercalation. Compared with monovalent metal ions, Ca²⁺ significantly enhances the stability of aptamer-OTA complexes. In addition, the CD spectra showed that Ca²⁺ and Mg²⁺ induced the formation of a hybrid G-quadruplex structure between aptamers and OTA. Molecular docking demonstrated that hydrogen bonds were formed between the aptamer bases DG-5 and DG-27 and OTA, with a dissociation constant (K_d) of 725 nmol/L. In summary, this study provides a systematic analysis of how solution conditions influence the interaction between aptamers and OTA, offering a theoretical basis for advancing the application of aptamers in biosensors.