A classic problem in preservation is the microbes can grow in low-moisture foods. In this paper, the water sorption, and thermodynamic properties of glucose/WPI solid matrices were measured, while their molecular mobility was analyzed and associated with the microbial growth ofD. Hanseniiat variousawand 30 °C. Although the sorption isotherms,Tg, and relaxation processes of studied matrices were affected byawand WPI, the microbial growth showed highly dependent on water mobility rather thanaw. Hence, we introduced water usability (Uw), derived from the mobility difference between system-involved water and liquid pure water explicating from the classical thermodynamic viewpoint, to describe the dynamic changes of water mobility in glucose/WPI matrices. Despite toaw, the yeast growth rate was enhanced at highUwmatrices concomitantly with a rapid cell doubling time. Therefore, the proposedUwprovides a better understanding of the water relationships of microorganisms in food preservation.