Okadaic acid (OA) is a typical marine toxin with strong toxicity causing diarrheic shellfish poisoning (DSP). Aptamers show great advantages in toxin detection and attract increasing attentions in the field of food analysis. In this study, a label-free col- orimetric aptasensor was constructed for visual and rapid detection of OA in shellfish. To exploit the binding capability of the anti- OA aptamer, the inherent molecular recognition mechanism of aptamer and OA was studied, based on molecular docking, fluorescent assay, and biolayer interferometry. Consistent results showed that the stem-loop near the 3\' terminal of the aptamer exhibit dominate binding capacity. Based on the revealed recognition information, the aptamer was thus rationally utilized and combined with AuNPs and cationic polymer polydiallyl dimethyl ammonium chloride (PDDA) for the development of the label-free colorimetric aptasensor, in which the 3\' terminal was thoroughly exposed to OA. The aptasensor provided robust performance with a linear detection range of 100 – 1200 nmol L–1, a limit of detection of 41.30 nmol L–1, recovery rates of 91.6％ – 106.2％, as well as a high selectivity towards OA in shellfish samples. The whole detection process can be completed within 1 h. To our best knowledge, this is the first time that the anti-OA aptamer was thoroughly studied, and a label-free colorimetric aptasensor was rationally designed in this way. This study not only provides a rapid detection method for highly sensitive and specific detection of OA, but also serves as a reference for the design of efficient aptasensors in the future.