Dimethyl sulfoxide is one of the most popular cryoprotectants in cryobiology. It is crucial for the successful cryopreservation to quantitatively analyze the cryoprotective properties of dimethyl sulfoxide. In this study, the osmolality and the unfrozen water content of the aqueous solution of dimethyl sulfoxide are determined using a Perkin-Elmer Diamond DSC. For precisely calculating the unfrozen water content, this study takes into consideration the effect of dilution on the enthalpy of fusion in the solution which is a commonly neglected factor. The osmotic virial representation and the quantitative relationships between the unfrozen water content and the solution composition are reported. The experimental results show that the aqueous dimethyl sulfoidde solution has a greatly depressed freezing point and a large osmolality. Moreover, the water-blocking ability of dimethyl sulfoxide weakens when the solution is more concentrated. The results in this study will enlarge the database of physical and chemical properties of dimethyl sulfoxide and reinforce our understanding of the cryoprotective mechanisms of dimethyl sulfoxide in aqueous solutions.