Objective Biosensors with multilayer biomedia are widely applied in various fields, and quantitative characterization of biosensors is still a problem for the development of sensors. This study is to quantitatively characterize the electrical properties of multilayer biomedia.Methods Combined with conformal mapping theory, the quantitative characteristics of biosensors are explored based on electrical impedance spectroscopy for clarifying the law of influence on impedance, and this study provides a basic theory for the characterization of biosensors. The impedance (Z^*) of each biomedia layer is extracted, and the simulation and calculation are executed to study the correctness.Results An experimental system has been established, results show that the impedance (Z^*) of the detection area continues to rise from the frequency (f) = 0.1 MHz to f=50.0 MHz in the coating process. This trend is explained that the solution in the original detection area is covered by the coating of biological medium with different dielectric properties, resulting in a decrease in the conductivity of the detection area and an increase in the impedance. Theoretical calculation results and simulation results show a great agreement with experimental results.Conclusion This study confirms that the multilayer biosensors are able to be quantitatively characterized based on electrical impedance spectroscopy and conformal mapping, which has certain practical value for the further development of biosensors.