By taking advantage of the intrinsic fluorescence of ochratoxin A (OTA), we present a fluorescence anisotropy approach for rapid analysis of the interactions between OTA and aptamers. The specific binding of OTA with a 36-mer aptamer can induce increased fluorescence anisotropy (FA) of OTA as the result of the freedom restriction of OTA and the increase of molecular volume, and the maximum FA change is about 0.160. This FA approach enables an easy way to investigate the effects of buffer compositions like metal ions on the affinity binding. FA analysis shows the interaction between OTA and aptamer is greatly enhanced by the simultaneous presence of Ca(2+) and Na(+), while the binding affinity of aptamer decreases more than 18-fold when only Ca(2+) exists, and the binding is completely lost when Ca(2+) is absent. Crucial region of the aptamer for binding can be mapped through FA analysis and aptamer mutation. The demonstrated FA approach maintains the advantages of FA in simplicity, rapidity, and robustness. This investigation will help the development of aptamer-based assays for OTA detection in optimizing the binding conditions, modification of aptamers, and rational design.