To quickly and intuitively detect the small offset of periodic structures, a 4f optical system-based method for detecting the small offset of periodic structures is proposed. Firstly, the VirtualLab Fusion optical simulation software is used for the theoretical study to formulate the model of periodic microstructure with preset offset, construct the optical transfer function, and obtain the image plane magnitude map corresponding to the periodic microstructure by using the 4f spatial filtering method. The analysis shows that in periodic structures of transparent substrates, regardless of size, if the offset is within 80% of the spacing between adjacent feature sizes, the fitted amplitude change is linear in relation to the microstructure offset and the location of the amplitude change is consistent with the location of the microstructure offset. The experimental system is established based on the simulated optical system parameters, and experimental results are consistent with the simulation. The system can achieve a measurement field of view of 3. 4 mm×2. 6 mm and a resolution of 5 μm, which is capable of real-time and rapid displacement or defect detection of periodic structural materials.