The permeation of riboflavin and insulin through poly(vinyl alcohol) (PVA) and chitosan blend membrane was conducted. The permeability coefficients of both solutes through the crosslinked PVA and chitosan blend membrane were in the order of 10−6−10−7 cm3 cm/cm2s and showed a pH dependence. The pH‐dependent permeation behavior was discussed in terms of water content and water structure inside of the swollen membrane. Riboflavin and insulin were presumed to permeate through the free water region in the swollen blend membrane. The DSC thermograms of these membranes indicated that the content of free water and the amount of freezing bound water increased with the water content in the membrane. The greater permeation rate of solutes in acidic solution rather than in neutral solution was due to an increase in both water content and the amount of free water and freezing bound water.