Abstract: With the development of inland waterway transportation, the demand for the construction of ultra-high-head ship locks is increasing day by day, and the contradiction between filling/emptying efficiency and vessel mooring safety is becoming increasingly prominent. The maximum hydraulic head of the navigation structure studied in this paper reaches 81.6 m, with a proposed layout of a two-level ship lock system consisting of an upstream first-level lock, a navigation tunnel, and a downstream second-level lock. The effective dimensions of both lock chambers are 190 m × 23 m × 4.7 m (length × width × sill depth). The design heads for filling the first-level lock and emptying the second-level lock both reach 40.80 m, exceeding the current highest water head of a single-level ship lock in China, which is 40.25 m at the Datengxia Ship Lock. To prevent large volumes of water from entering the navigation tunnel during the filling and emptying processes—which could affect vessel navigation within the tunnel—the system employs high and low regulating pools coupled with the two lock chambers to achieve large-volume water regulation and transfer between the upstream and downstream chambers. The unique configuration of two-level ship locks with high and low pools and an ultra-long water conveyance culvert presents significant hydraulic challenges for the design of the lock filling/emptying system and valve control. Based on the operation mode of a two-level dispersed ship lock system with high and low regulating pools, this study selects an appropriate filling/emptying system and determines the detailed dimensions of its components. While the filling process of the first-level ship lock is essentially identical to that of a conventional single-level ship lock, the filling process of the second-level lock involves water supply from both the low and high pools, making the hydraulic process and its impact on vessel mooring conditions significantly more complex. Thus, this study establishes a 1∶30 physical model of the high and low pools and the second-level ship lock, and conducts an experimental analysis of the hydraulic characteristics and vessel mooring conditions of the filling/emptying system of the second-level ship lock. The comprehensive analysis recommends that, when the low and high pools supply water to the second-level ship lock, the filling valves should be opened continuously for 4.0 minutes and closed for 2.0 minutes. The maximum filling flow rate in the lock chamber is 419.56 m³/s. The recommended valve operation method can control the filling time within 17.47 minutes. This conclusion confirms that, by using the recommended filling-valve operation method, the layout of the two-section filling/emptying system within the lock chamber, combined with different-sized branch openings in different zones, can effectively improve flow distribution within the chamber and ensure vessel berthing safety. The emptying valves for the second-level ship lock should be opened continuously for 9 minutes, and the lock emptying time can be controlled within 14.34 minutes. The maximum emptying flow rate in the lock chamber is 467.95 m³/s. In actual engineering projects, the second-level lock chamber can close the emptying valve within 2 minutes when the remaining head in the chamber is approximately 1.90 m. Additionally, measures such as opening the inverted arch gates when the water levels inside and outside the chamber are equal can be adopted to reduce the head-drop value of the second-level chamber. The research results of this paper can provide key parameters and a theoretical basis for the engineering design of this ultra-high-head ship lock and have important reference value for the design and construction of high-head navigation structures in western China.