The crystalline form A of Bimatoprost of formula I, its preparation method and use are provided. There are characteristic peaks where diffraction angles 2θ are 3.2±0.2°, 5.5±0.2°, 11.4±0.2°, 16.7±0.2°, 17.6±0.2°, 19.9±0.2°, 20.8±02°and 22.8±0.2° in the X-ray powder diffraction pattern of the crystalline form A.