OBJECTIVE: Since Augmented Reality (AR) and 3D visualization have proven to be of great significance to the safety and effectiveness of surgical outcomes, will autostereoscopic 3D AR without glasses bring new opportunities for surgical navigation of laparoscopic surgery? METHODS: We used the CPD-based deformation registration algorithm and the proposed virtual view generation algorithm, realizing a deformable autostereoscopic 3D AR navigation framework for laparoscopic surgery. The depth perception and user experience of the 3D AR navigation were evaluated compared with the 2D AR display using an in-vitro porcine heart and offline clinical partial nephrectomy laparoscopic images. RESULTS: The autostereoscopic 3D AR allowed participants to have a more consistent spatial perception as well as a shorter measuring time than 2D AR with significant difference of p < 0.05. It can also improve relative depth perception for smaller distance separation of objects < = 3.28 mm. However, the autostereoscopic 3D AR perceived a worse experience compared to 2D AR in the user experience. CONCLUSION: Autostereoscopic 3D AR shows a more efficient and robust sense of spatial scale than 2D AR with better potential to shorten the operating time and improve surgical outcomes than 2D AR, but image blur and distortion are issues that must be solved to improve the perception effect. High precise registration and high fluency visualization requirements could make autostereoscopic 3D AR navigation for soft tissue more challenging. SIGNIFICANCE: Our work lays a theoretical foundation for the further development of laparoscopic surgical navigation.