The multivalent effect is often used to engineer microfluidic affinity interfaces to improve the target separation efficiency. Currently, no design rules exist for thermodynamic and kinetic tuning of properly joining multiple ligands. Herein, we developed a thermodynamic and kinetic modulating strategy of the microfluidic affinity interface via a merit-complementary-heteromultivalent aptamers functionalized DNA nanoassembly. Our strategy is built on the two types of identified aptamers that bind to distinct sites of EpCAM. The aptamer binding of one type is more rapid but less tight, while the other is opposite. By assembling the two types of aptamers together with a tetrahedral DNA framework, we fully exploited these aptamers’ merits for tight and rapid recognition of EpCAM, leading to target cell capture with high efficiency and throughput. Our strategy provides a perspective on engineering multivalent recognition molecules through thermodynamic and kinetic tuning.