These days, multiplex assay with diverse functions on a single chip has become more and more imperative for biological cell research. Multipoint and multistep manipulation for single cells on a chip plays a significant role for cell characterization, immunoassays, and rare cell isolation, etc. In this article, a novel dielectrophoresis (DEP)-based manipulation method is proposed to flexibly move and position cells on a chip via applying various electrical signals onto microelectrodes. By modulating phases and amplitudes of alternating current (ac) signals applied onto the microelectrodes, single cells can be controllably moved from one position to another along diverse directions on a chip. Quantitative analysis is conducted for position and direction controls via simulation, which are validated through experiments. With this flexible manipulation method, single-cell biophysical parameters can be estimated in situ by moving the cell, and as an example, single HeLa and MCF-7 cells are measured. This method allows an efficient and flexible transportation of single cells in lab-on-a-chip systems and provides a fundamental platform for multioriented and multipoint manipulation. The chip is easy to scale up by means of array design for more multifunctional cell assays.