Objective: To evaluate the accuracy of a 3D-printed template used to assist the preparation of tibial tunnel in reconstruction of anterior cruciate ligament (ACL). Methods: Twenty healthy adult cadaveric knees were scanned by computed tomography(CT) and magnetic resonance imaging (MRI). The knees were from 11 males and 9 females who had died at an average age of 36 years (range, from 27 to 68 years) and from 8 left and 12 right sides. Individualized 3D reconstruction models of the knee joint were established based on their imaging data. According to the anatomic footprints of the virtual tibial tunnel, 20 individualized navigation templates were designed and printed by 3D printing. The templates were used to assisst preparation of tibial tunnels in the ACL reconstruction for the 20 cadaveric knees. After operation, CT scanning was conducted again to compare the corresponding postitions between the preoperative virtual tunnel and the postoperative actual tunnel. The positions of the tibial tunnel were described by the Tsukada method. Results: The ratio of the distance between the tunnel outlet center and the medial edge of the tibia to the distance between the median and lateral edges of the tibial plateau was 49.7%±2.1% for the preoperative virtual tibial tunnel and 48.8%±2.8% for the postoperative actural tunnel, showing no significant difference between them (t=1.971, P=0.063). The ratio of the distance between the tunnel outlet center and the anterior edge of the tibia to the distance between the anerior and posterior edges of the tibial plateau was 41.3%±1.3% for the preoperative virtual tibial tunnel and 40.3%±3.7% for the postoperative actural tunnel, showing no significant difference between them(t=1.494, P=0.152). Conclusion 3D printing can be used to design and manufacture a navigation template which can accurately assist preparation of tibial bone tunnel in ACL reconstruction.