The piezoelectric biosensor have been widely used in ultra-small mass detection of biomolecular, based on PZT piezoelectric material can create a variety of compositions geometrically; it could widely develop a high-frequency resonator and measure the change of the slightest mass while improve the limited detection simultaneously. Therefore, the piezoelectric biosensor of this study was fabricated by a spin-coating method and backside etching process for improving the characteristic of piezoelectric biosensor. The result exhibited that the 250 µm × 250 µm working size has the most favorable piezoelectric characteristic. The tunability was approximately 38.56 % and it showed that reducing the substrate thickness could obtain a clear resonance signal in a range of 60 to 380 MHz. In theory calculated for gravimetric sensing, it could achieve 0.1 ng sensing sensitivity. In gravimetric sensing, the sensing range was between 50,000~100,000 CFU/ml. Sensing range was lower in clinical urinary tract infection (100,000 CFU/ml), thus demonstrating its usefulness for preventive medicine. It can understand the piezoelectric sensor of this study has potential application in the future for biomedical gravimetric sensing.