Objective To calculate the pre-stretching of the microscopic components of the aortic wall under physiological homeostasis by considering a three-dimensional (3D) residual stress field. Methods The aortic wall was simplified into a double-layer ideal circular tube, and the 3D residual stress field of the vascular wall was calculated based on a 3D expansion angle experiment. Then, the in vivo stress distribution characteristics under mean blood pressure and the pre-stretching of each microscopic constituent of the vascular wall under a physiologically steady state were obtained. The inverse problem was constructed according to the internal pressure-radius relationship measured in vivo. Physiological homeostasis of the aorta was considered the reference state, and inversion identification of the material parameters of the aorta in vivo was realized while integrating the three residual stress fields. Results When the residual stress was not considered, the mean stress of the middle membrane was greater than that of the outer membrane. When residual stress was considered, the outer membrane bore more stress than the middle membrane, and the outer membrane protected the middle membrane. The pre-stretching of the middle film with residual stress is lower than that without residual stress, whereas the pre-stretching of the outer film is higher than that without residual stress. Moreover, the pre-stretching of the outer membrane collagen fibers was greater than that of the middle membrane collagen fibers. The in vivo calculations of the material parameters of the aorta were performed using physiological homeostasis as the reference configuration, and the proportion of each component was consistent with the experimental results. However, the proportion of elastin in the outer membrane was significantly overestimated when the non-stress configuration was used as the initial configuration, which was inconsistent with the experimental results. Conclusions Residual stress significantly influences the pre-stretching and physiologically steady mechanical states of the microscopic components of the aortic wall. Therefore, it is necessary to fully consider the influence of residual stress to establish the physiologically steady state of the aortic wall accurately. Furthermore, it is also necessary to fully consider the 3D characteristics and layer specificity of residual stress in the in vivo identification of material parameters.