For the automatic and non-destructive picking of the delicate and fragile brown mushroom, the biological and mechanical characteristics of the brown mushroom were firstly analyzed, the constraint equations of grasping force at pulling-off and twisting-off modes were given, and then the twisting-off picking mode was selected as an optimal method. Secondly, by using ANSYS, a finite element analysis software, the static analysis of the flexible finger grasping mushroom was carried out to give the relationships between the grasp force and the flexible finger knots, the diameter of brown mushroom, air pressure. The genetic algorithm was used based on the established evaluation function to obtain the optimal structure of the flexible gripper with three fingers and four knuckles, and the optimal air pressure of 18.65kPa for grasping. A flexible gripper with three fingers and four knuckles was developed based on the above study. And the brown mushroom picking experiment was carried out, and the results showed that the successful grasping force was reduced to（2.4±0.3)N compared with that of the rigid gripper with (5.6±0.7)N. Moreover, the surface and interior of the grasped mushroom by the developed flexible gripper were intact, while the surface and interior within 5mm depth of the grasped mushroom were damaged by using a rigid gripper. The research result showed that the designed flexible gripper with three fingers and four knuckles was feasible for brown mushroom picking automatically and non-destructively.