Abstract: For the feasibility study of microfocus X-ray phase contrast imaging in double and multi-layer plastic microsphere imaging and analysis, the images with clear characteristics of the double-layer plastic microsphere were obtained by choosing suitable experimental parameters on the consideration of imaging magnification, resolution, contrast and other factors, and based on in-line X-ray phase contrast imaging technique. Characteristic parameters of the double-layer microsphere, including inner layer thickness of (10.5 ± 0.6) μm, outer layer thickness of (9.2 ± 0.7) μm and external diameter of (273.3 ± 1.0) μm, were obtained by the use of segmentation methods, such as the Laplacian of Gaussian edge detection used to get the edges of each layer.