The composition of the ship's ballast water is complex and contains a large number of microalgae cells, bacteria, microplastics, and other microparticles. To increase the accuracy and efficiency of detection of the microalgae cells in ballast water, a new microfluidic chip for continuous separation of microalgae cells based on alternating current dielectrophoresis was proposed. In this microfluidic chip, one piece of 3-dimensional electrode is embedded on one side and eight discrete electrodes are arranged on the other side of the microchannel. An insulated triangular structure between electrodes is designed for increasing the inhomogeneity of the electric field distribution and enhancing the dielectrophoresis (DEP) force. A sheath flow is designed to focus the microparticles near the electrode, so as to increase the suffered DEP force and improve separation efficiency. To demonstrate the performance of the microfluidic separation chip, we developed two species of microalgae cells (Platymonas and Closterium) and a kind of microplastics to be used as test samples. Analyses of the related parameters and separation experiments by our designed microfluidic chip were then conducted. The results show that the presented method can separate the microalgae cells from the mixture efficiently, and this is the first time to separate two or more species of microalgae cells in a microfluidic chip by using negative and positive DEP force simultaneously, and moreover it has some advantages including simple operation, high efficiency, low cost, and small size and has great potential in on-site pretreatment of ballast water.