A theoretical model is developed to examine the effect of bending rigidity on measuring the adhesion work between a flat punch and a flexible membrane by pull-off test. The validity of this model is certified by the theoretical analysis. It can be used in the future experiments to analyze the experimental data when the bending rigidity of the membrane cannot be ignored. From our quantitative analysis, the effect is remarkable and sensitive to the bending rigidity. The effect of bending rigidity should not be neglected unless the membrane is very soft or the membrane is very thin. The elastic modulus of membrane should be less than 1.6MPa when the thickness of the membrane is less than 10μm. Moreover, it is obviously that the thickness plays a key role in the bending rigidity.