Accurately obtaining information on the heterogeneity of CTCs at the single-cell level is a very challenging task that may facilitate cancer pathogenesis research and personalized therapy. However, commonly used multicellular population capture and release assays tend to lose effective information on heterogeneity and cannot accurately assess molecular-level studies and drug resistance assessment of CTCs in different stages of tumor metastasis. Herein, we designed a near-infrared (NIR) light-responsive microfluidic chip for biocompatible single-cell manipulation and study the heterogeneity of CTCs by a combination of the lateral flow microarray (LFM) chip and photothermal response system. First, immunomagnetic labeling and a gradient magnetic field were combined to distribute CTCs in different regions of the chip according to the content of surface markers. Subsequently, the LFM chip achieves high single-cell capture efficiency and purity (even as low as 5 CTCs per milliliter of blood) under the influence of lateral fluid and magnetic fields. Due to the rapid dissolution of the gelatin capture structure at 37 °C and the photothermal properties of gold nanorods, the captured single CTC cell can be recovered in large quantities at physiological temperature or released individually at a specific point by NIR. The multifunctional NIR-responsive LFM chip demonstrates excellent performance in capture and site release of CTCs with high viability, which provides a robust and versatile means for CTCs heterogeneity study at the single-cell level.