Recent studies have suggested that the deformability of cells can be an effective biomarker to indicate the progression of diseases. Because of the obstacle of cellular heterogeneity, it was very difficult to make new cells stretch under the reported experimental conditions. In this paper, a cell electro-stretching method based on dielectrophoresis-coupled microfluidic chip was optimized and the effect of doxorubicin (DOX) on stiffness of leukemia NB4 cells was investigated using the established method. Firstly, positive dielectrophoresis (pDEP) method for cell capturing was optimized and validated, and the results demonstrated that it was easy to obtain pDEP effect of cells if the conductivity of cell suspension (σ_e) was so low as to make “cross-over” frequencies appear, meanwhile the frequency of the E-field f was adjusted in the range of the “cross-over” frequencies from Low-f₀ to High-f₀. Subsequently, three parameters affecting cell stretching were systemically discussed, and the results suggested that it was a prerequisite for cell electro-stretching that the conductivity of cell suspension (σ_e) was lower than that of cell cytoplasm (σ_c). In addition, increased voltage or decreased frequency of the E-field can also prompt cell stretching. Finally, the correlation between cell mechanical behavior and cell functional variation of NB4 cells was investigated via the optimized cell electro-stretching method, and the results showed that DOX treatment could make NB4 cells produce apoptosis and stiffen. Taken together, this study provides a novel cell electro-stretching method for the simple and efficient detection of cell deformability.