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Aiming at the problems of the existing plant stalk clamping device, such as clamping injury, clamping stroke and unadjustable clamping force, a plant stalk flexible clamping device was designed. The built-in spring of the clamping hand can realize the flexible clamping of plant stalk, and double adjust the clamping force and clamping stroke, so as to better meet the demands of plant stalk clamping in the fields of mechanics experiment, grafting and cutting test. Based on the rigid-flexible coupling simulation model of the clamping device, the multi-factor dynamic comparison simulation and test analysis were carried out, including clamping force, clamping stroke and spring stiffness. The sensitivity and stability analysis were carried out with the spring stiffness as the design variable to study the influence of different spring stiffnesses on the clamping performance of the flexible clamping device. The simulation and experiment results showed that the maximum clamping diameter of the device was 68.8mm, and the clamping stroke, speed and clamping force can be controlled smoothly based on the accurate clamping force model of the clamping device. With the increase of spring stiffness, the clamping force was gradually increased, and the clamping stability would be increased. Comprehensive comparison showed that when the spring stiffness was 10N/mm, it can meet the stable clamping demand of plant stems when the device applied 400N cutting force, and the flexible clamping force had little fluctuation, which can effectively avoid damage to plant stems.