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The path trajectory planning of orchard spray robot affects the smooth line of robot driving route and the reliability and smoothness of the driving process which needs more comprehensive consideration and more comprehensive planning. Aiming at the problems that the reference trajectory at the turn is not smooth enough and the curvature is large in the path planning of orchard spray robot, a trajectory optimization method of cubic non-uniform B-spline curve for orchard spray robot based on kinematics multiple constraints of orchard spray robot was proposed. The prior map was used to obtain the position information of the tree rows, and the path points between the rows were fitted to ensure that the orchard spray robot driving on the center line of the tree row met the requirements of the spray operation. The objective function of minimizing the path curvature was constructed by considering the minimum turning radius, the constraint of the first and end points, the delay constraint of the steering mechanism, and the continuity of curvature. The curve parameters to be optimized were solved by the optimization algorithm, and the global path that met the driving requirements of the orchard spray robot was generated. Finally, the pure tracking algorithm was used to verify the driving accuracy of the robot. The simulation and test results showed that the maximum curvature of the planned trajectory was 0.31m-1, and the average curvature was 0.15m-1, which met the driving requirements of the orchard spray robot. The average error of the trajectory tracking driving was 0.225m, and the mean square error was 0.031m, which met the requirements of the orchard spray robot for driving accuracy when spraying in the orchard.