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To improve the positioning accuracy of tunnels for anterior cruciate ligament (ACL) reconstruction, we proposed an ACL reconstruction robotic positioning system based on anatomical characteristics. The system includes a preoperative path planning system, an intraoperative path planning system, and a navigation and positioning system. Brahmet line, anterior, and posterior cortical lines are used for registration of preoperative computed tomography (CT) images and intraoperative X-ray images. A new calibrator of C-arm is applied to establish the mapping between medical images and surgical space. Tunnels for ACL reconstruction can be built anatomically by the robot. The accuracy of the path planning system is 1.73 mm in the four dry bones experiments and 2.17 mm in the two cadaver experiments. The accuracy meets the accuracy requirement of ACL construction surgery.