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According to the characteristics of potato tissue culture seedlings and their culture environments, a picking seedling machinery which can pick seedlings out of bottle without damage in one time was designed. It included a manipulator of mother bottle and a seedling grasper. The seedling grasper was used for holding the root of potato seedlings stationarily, meanwhile the manipulator moved backward with the mother bottle. The potato seedlings and the bottle moved relatively and then most part of the seedlings’ stems were out of the bottle, which provided good basis for the next operation in the automated micropropagation. The key parameters of picking seedling machinery were analyzed. In order to find the effect of the distance between the two pairs of claws, the number of each bottle, speed of manipulator and the swing frequency of mother bottle on the result of picking seedlings, orthogonal experiments were done. The index was inclination of culture medium after picking seedlings. The result showed that the distance between the two pairs of claws exerted the greatest effect on the inclination of culture medium, and other factors such as the number of each bottle, speed of manipulator and the swing frequency of mother bottle had no significant effects. As the distance between the two pairs of claws was 26 mm, the number of potato seedlings in each bottle was 45~55, the speed of manipulator was 25 mm/s, and swing frequency of mother bottle was 2 Hz, the minimum inclination of culture medium can be obtained by using the designed seedling mechanism for potato tissue culture seedlings.