Aptamer-functionalized microfluidic interfaces hold great potential for liquid biopsies owing to their programmable nature. However, most previous studies have focused on the development of multivalent aptamers to improve binding affinity, while ignoring aptamer orientation on microfluidic interfaces, resulting in suboptimal accessibility and affinity. Herein, we report a Cubic DNA Nanostructure (CDN)-programmed strategy to precisely control the orientation and valency of Aptamer on a microfluidic interface (CDN-Apt-Chip) for enhancing the capture and release of circulating tumor cells (CTCs). We demonstrate that the ordered orientation and multivalent configuration can synergistically increase the binding affinity of aptamers towards CTCs. By using CDN-Apt-Chip, we successfully isolated CTCs from the peripheral blood of T-cell leukemia patients, and discriminated T-cell leukemia patients from healthy volunteers. Furthermore, the captured CTCs were nondestructively released via nuclease treatment. We then performed T cell receptor sequencing on the released cells to demonstrate the compatibility with downstream analysis. Overall, this study provides a new paradigm for interface regulation of functional microfluidic chips and advances the clinical translation of aptamer-based liquid biopsy.