Rapid detection of okadaic acid (OA) in shellfish is crucial for practical application in food safety analysis. In order to establish a rapid, delicate detection scheme, an OA aptamer was utilized to quickly capture OA from the sample solution with polystyrene microspheres as solid phase carriers, and an inner-microchannel dam structure was designed to intercept the aptamer-functionalized microspheres to achieve the separation of OA for detection. Horseradish peroxidase (HRP) is utilized to catalyze the luminescence reaction of luminol-H2O2 solution. Through the direct competition for the aptamer between OA and OA-HRP, the rapid detection of OA can be achieved. The dynamic range of this detection method is 41.3–4.02 ng/mL, and the limit of detection (LOD) and lowest limit of quantitation (LOQ) are 12.4 pg/mL and 41.3 pg/mL, respectively. This miniaturized device enables rapid, ultrasensitive detection of OA, and demonstrates the merits of its field portability and low reagent consumption. The device can be deployed for on-site detection and analysis of marine biotoxins thereof.