Isolation of microalgal cells is as an indispensable part of producing biofuels for energy security and detecting toxic contaminants for marine routine monitoring. Microalgae live together with various microalgae naturally, and abundant samples need to be tackled in practical applications. Therefore, effective separation technologies need to be developed urgently to achieve high-throughput separation of various microalgae. Herein, we develop a reliable device to characterize the dielectric response of microalgae and sequentially separate various microalgae utilizing dielectrophoretic force in a bipolar electrode (BPE) arrayed device. First, by investigating the array width extension (AWE) effect on the electric- and flow-field distributions, we explore consequences of incidental electrohydrodynamic mechanisms and axial flow rate on the separation. Second, based on device performance on sample characterizations, we demonstrate this technology by separating microparticles in three- and five-channel devices. Third, we discriminate dead and live cells to explore its capability using the cell viability test and illustrate the AWE influence on the separation. Fourth, we characterize dielectric responses of different microalgae and separate C. vulgaris and Oocystis sp. Finally, we extended BPEs in length and developed an arrayed device for sequential separation of various microalgae, and this platform is successfully engineered in high-throughput isolation of C. vulgaris from complex samples. This technology presents good potential in addressing depleting fossil fuel and burgeoning environmental concerns due to its performance in the separation of microalgal strains from complex samples.