Metastasis caused by the entry of circulating tumor cells (CTCs) into the bloodstream or lymphatic vessels is a major factor contributing to death in cancer patients. Separation of CTCs and studies on CTC-drug interactions are very important for prognostic and therapeutic implications of metastatic cancer. In this study, an integrated microfluidic device for CTC separation through the combination of lectin and microstructure is presented. This microfluidic device and lectin concanavalin A were utilized for the separation of K562 cells in whole blood samples. The results showed that the separation efficiency can reach 84%, which is much higher than that of an experiment without concanavalin A treatment. To further demonstrate the feasibility of this microfluidic device application in sequential studies after target cells were separated, the interactions of K562 cells and an anticancer drug, cytarabine, were also examined. After 6 h on-chip treatment with cytarabine, the viabilities of K562 cells were 85.29, 77.05, and 40% for drug concentration levels of 0.25, 0.5, and 1.0 g/L, respectively. This system can facilitate the rapid and efficient in vitro investigation of CTC separation and CTC-related studies.