In order to measure the air-space thickness of Glan-Taylor prisms, effects of prisms on the single-mode Gaussian beam were analyzed. The result indicates that the transmitted beam light intensity shows periodical oscillation with change of incident angles on the prism’s end face. And the characteristics of the oscillation is related to the incident light wavelength, beam intensity distribution, prism structure angle and air-gap thickness. For the incident single-mode Gaussian beam with given wavelength, the structure angle can be measured exactly before the prism is agglutinated, so the air-gap thickness can be measured by analyzing the characteristics of the oscillation. The experiment is designed, and the periodical oscillation characteristics are measured. Using the computer program, average relative deviation of the experimental oscillating period measured and the theoretical one was calculated by changing the air-space thickness in theoretical calculation with the interval of 0.0001 mm. For the sample prism, the minimum average relative error is 4.35%, and the air-space thickness is 0.0143 mm.