It is difficult to accurately measure the volume of transdermally extracted interstitial fluid (ISF), which is important for improving blood glucose prediction accuracy. Skin resistance, which is a good indicator of skin permeability, can be used to determine the volume of extracted ISF. However, it is a challenge to realize in vivo longitudinal skin resistance measurements of microareas. In this study, a three-electrode sensor was presented for measuring single-point skin resistance in vivo, and a method for determining the volume of transdermally extracted ISF using this sensor was proposed. Skin resistance was measured under static and dynamic conditions. The correlation between the skin resistance and the permeation rate of transdermally extracted ISF was proven. The volume of transdermally extracted ISF was determined using skin resistance. Factors affecting the volume prediction accuracy of transdermally extracted ISF were discussed. This method is expected to improve the accuracy of blood glucose prediction, and is of great significance for the clinical application of minimally invasive blood glucose measurement.