Input design has a dominant role in developing the dynamic model of an autonomous underwater vehicle (AUV) through system identification. Optimal input design is the process of generating informative inputs that can be used to provide a good-quality dynamic model of AUV. In this paper, amplitude-modulated pseudo-random binary signal (APRBS) inputs are optimally designed in order to estimate the hydrodynamic derivatives of an AUV’s nonlinear dynamic model. The input controls are designed so as to minimize uncertainty in estimating hydrodynamic derivatives. The employed approach can design multiple inputs and apply constraints on an AUV system’s inputs and outputs. The genetic algorithm is utilized to solve the constraint optimization problem. The presented algorithm is used for designing the input signals of Hydrolab300 AUV, and the estimation obtained by these inputs is compared with that of zigzag maneuver. According to the results, the designed APRBS inputs improve the uncertainties that exist in estimating hydrodynamic derivatives better than zigzag inputs.