Cell dielectric properties, a type of intrinsic property of cells, can be used as electrophysiological biomarkers that offer a label-free way to characterize cell phenotypes and states, purify clinical samples, and identify target cancer cells. Here, we present a review of the determination of cell dielectric properties using alternating current (AC) electrokinetic-based microfluidic mechanisms, including electro-rotation (ROT) and dielectrophoresis (DEP). The review covers theoretically how ROT and DEP work to extract cell dielectric properties. We also dive into the details of differently structured ROT chips, followed by a discussion on the determination of cell dielectric properties and the use of these properties in bio-related applications. Additionally, the review offers a look at the future challenges facing the AC electrokinetic-based microfluidic platform in terms of acquiring cell dielectric parameters. Our conclusion is that this platform will bring biomedical and bioengineering sciences to the next level and ultimately achieve the shift from lab-oriented research to real-world applications.