This study verifies the effects of deployment parameters on the safe separation of Autonomous Underwater Vehicles (AUVs) and mission payloads. The initial separation phase is meticulously modeled based on computational fluid dynamics (CFD) simulations employing the cubic constitutive Shear Stress Transport (SST) k-ω turbulence model and overset grid technologies. This phase is characterized by a 6-degree-of-freedom (6-DOF) framework incorporating Dynamic Fluid-Body Interaction (DFBI), supported by empirical validation. The SST k-ω turbulence model demonstrates superior performance in managing flows characterized by adverse pressure gradients and separation. DFBI entails computationally modeling fluid–solid interactions during motion or deformation. The utilization of overset grids presents several advantages, including enhanced computational efficiency by concentrating computational resources solely on regions of interest, simplified handling of intricate geometries and moving bodies, and adaptability in adjusting grids to accommodate changing simulation conditions. This research analyzes mission payloads’ trajectories and attitude adjustments after release from AUVs under various cruising speeds and initial release dynamics, such as descent and angular velocities. Additionally, this study evaluates the effects of varying ocean currents at different depths on separation safety. Results indicate that the interaction between AUVs and mission payloads during separation increases under higher navigational speeds, reducing the separation speed and degrading the stability. As the initial drop velocities increase, fast transition through the AUV’s immediate flow field promotes separation. The core of this process is the initial pitch angle management upon deployment. Optimizing initial pitching angular velocity prolongs the time for mission payloads to reach their maximum pitch angle, thus decreasing horizontal displacement and improving separation safety. Deploying AUVs at greater depths alleviates the influence of ocean currents, thereby reducing disturbances during payload separation.