Rapeseed seedling transplanting is an innovative and efficient transplanting technique. However, falling off and high substrate loss have become the common problem facing rapeseed seedling transplanting fields and causing the transplanting quality of rapeseed seedling decrease. A clipping type seedling pick-up device was developed. According to the position requirements of the pick-up arm, the size relationship between the components of pick-up device was determined. The displacement equation and motion trajectory of pick-up arm were obtained by dynamic analysis. The optimal parameter combination was obtained based on Matlab optimization analysis, so as to reduce the horizontal spacing (ΔxE) and the height (ΔyE) of trajectory, which were as follows: the length of active side-stick was 75.10mm, the length of pick-up arm was 335.26mm, the length of negative side-stick was 100.42mm, and the length of frame was 171.32mm. And the optimization results were as follows: the horizontal spacing of trajectory was 173.20mm, and the height of trajectory was 29.56mm. The critical falling off equations of seedling block was established by dynamic analysis. The mechanical and physical properties of seedling block were obtained by experiment. The following conclusions were obtained through analysis: the minimum clamping force for pick-up device was 7.07N, and the diameter of the finger cylinder was 20mm. The virtual trajectory was obtained by ADAMS, and the actual trajectory was obtained by high-speed photography technology. After comparison and analysis, the error between the actual value, optimization result and simulation result were less than 3%, which verified the correctness of the mechanism design. The seedling pick-up was tested through the bench experiment, and the results were as follows: the success rate of seedling pick-up was 93.33%, the rate of seedling falling off was 2.86%, and the rate of substrate loss was 3.75%, which met the requirements of rapeseed substrate seedling pick-up and verified the feasibility of the mechanism design. The research result provided a reference for the design of pick-up device for rape substrate seedling transplanter.