One of the work kind modes of the multilayer piezoelectric biosensors is to detect changes in resonant frequency resulted from binding and hybridization of biomolecules. In this work, mechanical design issues of multilayer piezoelectric biosensor (consisting of Si, SiO2, PZT, Pt electrode and biolayer) with high sensitivity are investigated. Piezoelectric effect matrices are included in the finite element simulation model. Harmonic response analysis is employed to determine the geometry parameters of the multilayer biosensor and predict the piezoelectrical signals subjected to base vibration. The relationships between the dimensions of the multilayer and the resonance frequency, mass sensitivity, piezoelectrical response are studied. At the same time, the thermal mismatch effects on the mechanical and electrical characteristics of the multilayer biosensor are also investigated. It is shown that the frequency of the sensor increase by the thermal mismatch bending, while it is small in the application temperature range of the biosensors. Simulation results also showed that the multilayer piezoelectric biosensor can have a greatly enhanced minimum detectable mass density (MDMD) of 0.05 ng/cm2, as compared to 10 ng/cm2 from the quartz crystal microbalance (QCM).