An antibody-free approach for high purity and high throughput isolation of circulating tumor cells (CTCs) from human blood in a microfluidic chip is presented. High throughput continuous CTC isolation (>1.2 ml/hr) is achieved with a sustained dielectrophoresis (DEP) particle force normal to the continuous through-flow. The design allows continuous fractionation of cells into different downstream sub-channels based on the differences in their different critical DEP strengths/mobility on both sides of the cross-over. An enrichment factor of ∼105 and a recovery rate of ∼80% from a 0.001% CTC sample is achieved at a high throughput of 1.2 ml/hr. Compared to conventional physical methods, such as immunomagnetics-based methods and microfluidic biomarker-based methods, the isolation purity, recovery rate, throughput and viability of the proposed antibody-free chip demonstrated excellent capabilities, especially with regard to high enrichment at a high flow rate.