Tumor metastasis is attributed to circulating tumor cells (CTC) or CTC clusters. Many strategies have hitherto been designed to isolate CTCs, but there are few methods that can capture and gently release CTC clusters as efficient as single CTCs. Herein, we developed a three-dimensional (3D) scaffold chip with thermosensitive coating for high-efficiency capture and release of individual and cluster CTCs. The 3D scaffold chip successfully combines the specific recognition and physically obstructed effect of 3D scaffold structure to significantly improve cell clusters capture efficiency. Thermosensitive gelatin hydrogel uniformly coated on the scaffold dissolves at 37 °C quickly, and the captured cells are gently released from chip with high viability. Notably, this platform was applied to isolate CTCs from cancer patients' blood samples. This allows global DNA and RNA methylation analysis of collected single CTC and CTC clusters, indicating the great potential of this platform in cancer diagnosis and downstream analysis at the molecular level.