Single cell assays are being established using lab-on-a-chip technologies. Impedance provides an easy, non-invasive way to count, classify, and track cellular activity. For single cell impedance, a variety of microfluidic devices have been designed. Simple cell counting and label-free detection of various cell types and detection of cell morphological changes are few out of many potential applications. Devices that flow single cells and continuously record impedance data have also been developed. Fundamental analysis, diagnostics, and non-invasively probing cell activity at the single-cell level are all possible uses for this technology. A three-dimensional microfluidic chip has been developed for real-time and non-invasive impedimetric for cell counting. This study describes the fabrication and characterization of a microfluidic device for the impedance spectroscopy of biological cells. Key features of the device include all designing & fabrication processing for the formation of fluidic channels, coplanar metal electrodes for impedance measurements, surface modification, alignment, and irreversible bonding. In addition, new, improved microelectrode architecture is presented, which is used in the construction of impedimetric biosensors to improve the signal-to-noise ratio of biological cell electrical signature. The device uses a differential enumeration platform that integrates Coulter counting principles for cell counting. The heterogeneous population of leukemia cells (MV4-11) is calculated in a 3D miniaturized channel bonded with a new coplanar microelectrode configuration for proof of concept. The applications of these new microelectrode designs have provided unprecedented opportunities for the development of high-performance impedance biosensors. This impedimetric microfluidic chip has a high potential to develop a powerful platform for cancer research for cell proliferation and chemosensitivity monitoring.