Food safety is a major issue to public health and have attracted global attention. Fast, sensitive, and reliable detection methods for food hazardous substances is highly desirable. Aptamers which can bind to the target molecules with high affi nity and specifi city represent an attractive tool for the recognition of food hazardous substances, which play an important role in the development and application of new food safety detection technology. But current assays for characterizing small molecule-aptamer binding are limited by either the mass sensitivity or the size differentiation ability. Herein, we proposed a comprehensive method for assessing the dissociation equilibria of small molecule-aptamer, which is immobilized-free under ambient conditions. The design employs the Le Chatelier’s principle and could be used to effectively measure small molecule-aptamer interactions. ATP binding aptamer and anti-afl atoxin B1 aptamer were used as the model system to determine their affi nity, in which their dissociation equilibria measurements are in excellent close to their previous work. Due to the simplicity and sensitivity of this new method, we believe that it could be recommended as an effective tool for characterizing small molecule-aptamer interactions and promote the further application of small molecular aptamer in food safety.