The transplantation of Panax notoginseng seedlings are always relying on artificial， resulting in low operation efficiency and poor planting quality. Panax notoginseng seedlings may be easily intertwined with another, the existing separation device cannot effectively separate Panax notoginseng seedlings, the seedling separation device is a key component to achieve planting mechanization of Panax notoginseng. In order to obtain discrete and orderly seeding of Panax notoginseng seedlings, a kind of seedling separation device of Panax notoginseng seedlings transplanter based on roller serrated mechanism was designed. Based on theoretical calculation, the main components were designed, including spike, roter, roller, seedling feeding device and so on. The number of helix,length of rotor nail, length of the nail and the number of guide grooves were taken as the virtual testing factors, and virtual prototype model was created according to different levels parameter of structural by Solidworks, and then the models were imported into the discrete element simulation analysis software EDEM, the seedling variation coefficient and discrete index were used as index to launch single factor and orthogonal simulation experiments. The results showed that the optimum structure parameters were as follows: the number of spiral was 4, the rotor nail tooth length was 70mm, the roller nail tooth length was 65mm and the guide groove number was 3, at this time, the separating coefficient of variation of separation device was minimal and discrete exponent was the highest. Through the practical experiments, the simulation results were verified, and the results were consistent with the simulation results: the minimum coefficient of variation was 17.37%, the highest separation rate was 84.69%, and the broken rate was 7.5%. The research achieved the orderly and discrete separation of Panax notoginseng seedlings, which provided a reference for the further study of Panax notoginseng and other rhizome crops separation device.