For precision enhancement, many robotic systems for minimally invasive surgery (MIS) have been developed. However, a positioning arm that firmly fixes the initial position of surgical robot arm has not been researched in depth yet. Since a positioning arm is a device for positioning a surgical robots, it should be equipped with a robust locking mechanism against external force. Furthermore, as repositioning of the surgical robot occurs frequently before and during the operation, the positioning arm needs a mechanism to lock and unlock multi-degrees-of-freedom (MDoF) simultaneously to reduce the setup time. Therefore, we propose a novel MDoF locking mechanism applicable to a positioning arm for MIS. When the knob of the locking mechanism is rotated, the two joints are locked simultaneously by the movement of the friction pad and the locking block that is mounted inside the positioning arm. Considering the locking time and the locking torque, the design parameters of the locking mechanism were determined to be optimal. The proposed locking mechanism was implemented and verified on a positioning arm for MIS. The first experimental results showed that only the negligible clearance between the joints was generated during the locking and unlocking transition. These indicate that positioning of the surgical robot is precise when the proposed mechanism is applied. The second experimental results showed that the proposed locking mechanism has a higher performance than the conventional locking device, in terms of locking torque. These indicate that the proposed locking mechanism will enhance safety in a surgical environment.