The interaction between glycerol and water molecules is very complex, which displays different plasticization properties. In this paper, it studied the influence of glycerol-water mixture with different glycerol ratio on the thermal and rheological properties of starch. In water-rich mixture (glycerol ratio is less than 50%), its plasticization effect is similar as water, but will be impaired with the increase of glycerol. In glycerol-rich mixture (glycerol ratio is more than 90%), its lubrication and plasticization effect is similar as glycerol, and since glycerol can abstract lipid and destroy amylose-lipid complex, the peak M2 disappeared in DSC curves. In transition mixture, the mutual interactions reach maximal and less active hydroxy left, so the plasticization effect is the weakest, which is reflected by the highest loading peak and phase transition peak in torque-time curve by rheometer.