Buffer type, concentration, and pH value are important factors that may affect protein formulation, stability, bioreactivity, and other behaviors. When biosensing application factors are the issues, the effect of buffer liquidity on the performance of the sensor should be noted. In this work, we investigated the effect of the phosphate buffer (PB) solution on the vibrational characteristics of a high-frequency quartz crystal microbalance (QCM) with an inverted-mesa structure using solutions with various concentrations and pH values. Experimental results show that increasing the PB concentration and pH value and adding NaCl decreased the resonant frequency and quality factor. Hence, in selecting the buffer, one should not only consider the bioissues, but also the effect on the sensor when a QCM is used as the transducer.