Mass sensitive piezoelectric biosensors based on micro-electro-mechanical system (MEMS) fabrication techniques were developed for biomaterial detection. In this work, both 4-inch double-sided polished silicon wafer was used as support layers. The SiO2/Si3N4 layers were firstly grown on both sides of the wafer to serve as the etching mask and the potassium hydroxide solution (KOH) wet etching stop layer. The diaphragm chambers were then wet etched in KOH solution until the remaining thickness of silicon was about 50μm. The residual thin silicon layer could reduce the stress and maintain a good mechanical strength for the following fabrication processes. After that, the bottom electrode/piezoelectric layer/polyimide layer/top electrode was fabricated and patterned on the other side of the wafer successively. Then the residual thin silicon layer in the chambers was etched away and a Au layer with 50nm thickness was sputtered on the bottom of the chambers as the immobilization surface for biomaterial specific adsorption. The square sensors with different length of sides were designed to investigate the size effect on the resonance performance. The resonant frequency shifts could be measured and the corresponding mass of the attached biomaterial could be calculated. The piezoelectric diaphragms sensors have a potential application for biomaterial detection.