The topographic features at the cell–material biointerface are critical for cellular sensing of the extracellular environment (ECM) and have gradually been recognized as key factors that regulate cell adhesion behavior. Herein, a well-defined nanostructured biointerface is fabricated via a new generation of mussel-inspired polymer coating to mimic the native ECM structures. Upon the bioinert background presence and biospecific ligands conjugation, the affinity of cancer cells to the resulting biofunctional surfaces, which integrate topographic features and biochemical cues, is greatly strengthened. Both the conjugated bioligand density, filopodia formation, and focal adhesion expression are significantly enhanced by the surficial nano-features with an optimized size-scale. Thus, this nanostructured biointerface exhibits high capture efficiency for circulating tumor cells (CTCs) with high sensitivity, high biospecificity, and high purity. Benefiting from the unique bioligands conjugation chemistry herein, the captured cancer cells can be responsively detached from the biointerfaces without damage for downstream analysis. The present biofunctional nanostructured interfaces offer a good solution to address current challenges to efficiently isolate rare CTCs from blood samples for earlier cancer diagnosis.