In this paper, a double closed-loop backstepping controller is designed for 3D trajectory tracking of a turtle-inspired amphibious spherical robot suffering from problems that include model uncertainties, environmental disturbances, and unmeasured velocity. The proposed controller scheme tackles three primary challenges: the differentiation explosion of the traditional backstepping method, unmeasured velocity, and the consideration of lumped disturbances. Beginning with an outer-loop backstepping controller, a virtual feedback variable is constructed to simplify the design of the backstepping controller. Meanwhile, to avoid the problem of differentiation explosion, tracking differentiators (TDs) are utilized to estimate the differentiation of the desired velocity in an inner-loop backstepping controller. Moreover, there are some uncertainty disturbances in the task of tracking the trajectory of a turtle-inspired amphibious spherical robot (TASR), such as the parameters of the hydrodynamic model and environmental disturbances. A linear extended state observer (LESO) is designed to estimate and compensate for the lumped disturbances. Furthermore, as the velocity states of the TASR are unmeasured, the LESO is also utilized to estimate the velocity states in surge, yaw, and heave degrees. Therefore, the TASR only needs to supply its position and orientation information for the trajectory tracking task. Note that this paper details both the design process of the proposed controller and a rigorous theoretical analysis. In addition, numerical simulations are conducted, and the results demonstrate the feasibility and superiority of the proposed method.