An accurate, easy setup, low-cost, and time-saving method for measuring glucose concentration was proposed. An all-grating-based glucose concentration measurement system contained moving-grating-based heterodyne interferometry and a grating-based self-align sensor. By combining the first-order diffraction lights from two separated moving gratings by a polarization beam splitter and creating S- and P-polarized light interference by an analyzer, the interference signal could be a heterodyne light source with a heterodyne frequency depending on the relative velocities of the two moving gratings. Next, a grating-based self-align sensor was used to make the optical configuration setup easy and accurate. Moreover, the sensor was deposited on GOx film to improve the measurement sensitivity and specificity for glucose. Finally, the phase change induced by the reaction of the sensor and glucose solutions was detected. The validity of this method was proved, and the measurement resolution can reach 2 mg/dL.