During the last decades, the growing interest for single-cell analysis has led to the creation of a number of microfluidic and lab-on-a chip (LOC) platforms for characterizing cellular samples. In that context label-free based platforms are minimally invasive and offer the notable advantages of reducing alteration of the analyzed sample and granting its re-employment. The study of intrinsic features of single cells independent from markers is commonly attained using electrical and mechanical-based techniques. Electrical-based techniques have been widely employed in LOC applications, both for characterizing and for manipulating cell samples. The translation of these approaches to single-cells necessitates microelectrodes that can be singularly addressed and arranged in a high-density topography. This thesis provides two fabrication solutions that comply with these requirements and allow to manufacture highly conductive vertical platinum microelectrodes with high aspect-ratio. According to the two processes reported, the three-dimensional (3D) cores of the electrodes are fabricated in SU-8 or in silicon respectively. These tridimensional structures are successively coated by a metal layer, after a passivation step in the case of silicon. The planar metal connections which singularly address the free-standing microelectrodes are patterned differently for the two approaches, respectively by lift-off and spray coating. Importantly, the 3D microelectrodes can be co-fabricated with microfluidic structures to obtain multiple active sites for single-cell analysis. In this thesis, in the framework of a collaboration with Ludwig Centre for Cancer Biology (Lausanne, Switzerland), the microelectrodes have been employed to detect activated T cells. The encouraging results pave the way to a new generation of microfluidic platform based on 3D microelectrodes to attain real-time and label-free monitoring of individual T cells to employ in immunotherapy.