Accurate diagnose of a disease in the early stage is critical to treat the disease properly. To this end, a multitude of biosensors with advanced technologies have been developed to detect the number of biomolecules precisely. In this work, we propose a method for extracting the Stern layer capacitance (Cstern) using the experimental data of silicon nanowire ion-sensitive field-effect transistors (ISFETs) to help improve the accurate detection of target molecules. The proposed method was applied to both pH and virus sensing scheme, and the Cstern value of pH and a virus were extracted as 32 and 26 mu F/cm2, respectively. These findings indicated that the extracted Cstern was affected by the size of the ion and protein, which also was verified by a computer-aided simulation. These insights would be useful in the development of charge-based ISFET biosensors.