In recent years, microfluidic dielectrophoresis (DEP) devices, as one of the most promising tools for cell and particle sorting and separation, are facing the bottleneck in the development of practical pfoducts due to the high-cost yet low yield device manufacturing via traditional microelectromechanical systems (MEMS) and the challenge of maintaining the cell viability during DEP treatment. In this paper, we demonstrate a facile, low-cost, and high-throughput method of constructing continuous-flow microfluidic DEP devices via screen-printing technology. The new device configuration and operation strategy not only facilitate cell and particle sorting and separation using 3D electrodes as sidewalls of microchannel but also improve cell viability by reducing the exposure time of cells to high electrical field gradients. Furthermore, we propose and validate a semiempirical formula with which to simplify the complicated calculation and plotting of DEP spectra. As a consequence, the optimal DEP parameters and crossover frequencies can be obtained directly using our devices instead of typical electrorotation method. To evaluate the performance of a screen-printed continuous flow microfluidic DEP device, a suspension containing polystyrene (PS) microspheres and erythrocytes is used as the biosample. Our results show that a high sorting efficiency (ca. 93%) with a high cell viability (hemolysis ratio of <4.8%) can be achieved, indicating the excellent performance and promising application of such devices for cell and particle sorting and separation.