The characterization of biological objects with microwave spectroscopy is getting increasing interests, as it is label-free and noninvasive. To perform their analysis, 2-port sensors are present in the literature, enabling only partial investigations of 3D biological samples, without taking their structural heterogeneity into account. Within this context, a 4-port microwave-based biosensor dedicated to microtissue characterization is proposed, in order to extend the sensing capabilities of microwave dielectric spectroscopy and provide electrical responses of 3D biological models subdivisions. An electrical model suitable for such a multiport device is established to extract the characteristics of the different sections of the 3D entity. The modeling methodology exploits the symmetry of the microwave component, while applying a common and differential modes approach derived from the measured 4 ports scattering parameters. After the mathematical validation of this approach, different elementary models are evaluated. Ethanol-based aqueous solutions are first used for their homogeneity within the fluidic channel. Polystyrene beads exhibiting two different diameter sizes are then numerically and experimentally investigated due to their 3D configuration and their uniform and known permittivity. This study demonstrates that the 4-port sensor and associated electrical model enable to consider electrical subdivisions of the 3D entity under test, based on the localization of the object on the different microwave electrodes. This constitutes the first step toward the analyses of complex and heterogeneous 3D biological models such as microtissues.