Posterior atlantoaxial pedicle screw fixation is an effective technique for atlantoaxial instability (AAI). However, because of the complex anatomy of the cranio-cervical junction, it remains challenging to insert atlantoaxial pedicle screw precisely and safely. A novel drill guide template was designed for atlantoaxial pedicle screw placement in this study. The purpose of this study is to quantitatively evaluate the accuracy of atlantoaxial pedicle screw placement using the novel drill guide template. Between June 2014 and September 2015, 32 consecutive patients with AAI were included. Using the Mimics v17.0 and 3-matic v9.0 softwares, a novel drill guide template with two location holes and guide rods was designed. All patients underwent posterior atlantoaxial pedicle screw fixation assisted by the novel drill guide template. After surgery, the entry point and directions of actual and ideal screw trajectories were measured and compared. All patients underwent surgery successfully assisted by the novel drill guide template. A total of 128 atlantoaxial pedicle screws were placed for 32 AAI patients. Postoperative CT scans showed two screws in atlas deviated medially from the pedicle cortex and entered the spinal canal about 1 mm but without symptoms. For both atlas and axis, there were no significant differences in entry point or directions between the ideal and actual screw trajectories (P > 0.05). Significant differences were found in preoperative and postoperative Japanese Orthopaedic Association score and Visual Analogue Score (P < 0.001). It is feasible to use the novel drill guide template for atlantoaxial pedicle screw placement. The accuracy of screw placement assisted by the novel template is high. More studies are needed to confirm the efficacy of this template.