The herbicide penetration on weed leaves was spatially analyzed by using chlorophyll fluorescent emission and machine vision technique. Velvetleaf and metribuzin were used as experimental materials in the study. The herbicide spray images were obtained by a combinaton of a fluorescent dye and a UV lighting system. The herbicide penetration was analyzed by means of detecting chlorophyll fluorescent emission under blue-green lighting. According to the experiment results, the number and the size of spray droplets decreased with coverage increasing. The herbicide penetrated mainly along leaf veins and the time for complete penetration over the whole leaf was approximately 100 minutes after herbicide spraying. When the coverage of herbicide droplets on the surface of leaves increased, the speed of herbicide penetration also increased. This study suggested a way of characerizing herbicide spatial penetration and distribution in leaves.