Dielectrophoretic (DEP) separation has been recognized as a practical tool in the separation of cells and particles for clinical diagnosis, the pharmaceutical industry and environmental monitoring. Assembly of particles and cells under DEP force is a common phenomenon and has an influence on their separation but has not been understood fully. Encouraged by these aspects, we developed a microfluidic device with a bipolar electrode array to investigate the assembly and separation of particles and cells at a large scale. First, we studied the assembly and evolution mechanisms of particles of one type under an AC electric field. Then, we investigated the interaction and assembly of multiple particles with dissimilar properties under DEP force. Depending on the development of microfluidic devices, we visualize the assembly process of yeast cells at the electrode rims and of polystyrene particles at the channel centers, and explore the influence of pearl chain formation on their separation. With increasing flow velocity from 288 to 720 μL h-1, the purity of 5 μm polystyrene particles surpasses 94.9%. Furthermore, we studied the DEP response of Scenedesmus sp. and C. vulgaris, and explored the influence of cell chains on the isolation of C. vulgaris. The purity of Scenedesmus sp. and C. vulgaris witnessed a decrease from 95.7% to 90.8% when the flow rate increased from 288 to 864 μL h-1. Finally, we investigated the extension of the electric field under chains of Oocystis sp. at the electrode rims by studying chain formation and capture of C. vulgaris, and studied its effect on cell chain length, recovered cell purity and cell concentration. When chains of Oocystis sp. were formed, the purity of C. vulgaris kept unchanged and the concentration decreased from 2793 cells per μL to 2039 cells per μL. This work demonstrates continuous DEP-based assembly and separation of particles and cells, which facilitates high-efficiency isolation of targeted cells.