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Current surgical methods to treat atlantoaxial instability pose potential risks to the surrounding blood vessels and nerves of operative approach. Therefore, more secure and highly effective methods are expected. This study sought to assess the biomechanical efficacy of a novel unilateral double screw-rod fixation system by comparing with traditional and emerging fixation methods in cadaveric models.Ligamentous cervical spines (C0-C7) from ten fresh cadaveric specimens were used to complete range of motion (ROM) test in their intact condition (control group), destabilization, and stabilization after different fixations, including unilateral C1-C2 pedicle screws (PS) with a screw-rod system (Group A), bilateral C1-C2 PS with screw-rod systems (Group B), unilateral C1 posterior arch screws (PAS) and C2 laminar screws (LS) combined with an ipsilateral paralleled C1-C2 PS-rod (Group C), and unilateral C1 PAS and C2 LS combined with an ipsilateral crossed C1-C2 PS-rod (Group D). After that, pullout strength test was performed between PS and PAS using ten isolated atlas vertebras.All fixation groups reduced flexibility in all directions compared with both control group and destabilization group. Furthermore, comparisons among different fixation groups showed that bilateral C1-C2 PS-rod (Group B), unilateral C1 PAS + C2 LS combined with an ipsilateral paralleled C1-C2 PS-rod (Group C) and unilateral C1 PAS + C2 LS combined with an ipsilateral crossed C1-C2 PS-rod (Group D) could provide a better stability, respectively, in all directions than unilateral C1-C2 PS-rod (Group A). However, no statistical significance was observed among Groups B, C, and D. Data from pullout strength test showed that both C1 PS (585 ± 53 N) and PAS (463 ± 49 N) could provide high fixed strength, although PS was better (P = 0.009).The surgical technique of unilateral C1 PAS + C2 LS combined with a ipsilateral crossed C1-C2 PS-rod fixation could provide a better stability than the traditional unilateral PS-rod fixation and a same stability as bilateral PS-rod fixation, but with less risk of neurovascular injury. Therefore, this new technique may provide novel insight for an alternative of atlantoaxial instability treatment.