Isobaric gas-tight hydrothermal samplers, with the ability to maintain pressure, can be used to keep in situ chemical and biological sample properties stable. The preloading pressure of the precharged gas is a major concern for isobaric gas-tight hydrothermal samplers, especially when the samplers are used at different sampling depths, where the in situ pressures and ambient temperatures vary greatly. The most commonly adopted solution is to set the preloading pressure for gas-tight samplers as 10% of the hydrostatic pressure at the sampling depth, which might emphasize too much on pressure retention; thereby, the sample volume may be unnecessarily reduced. The pressure transition of the precharged gas was analyzed theoretically and modeled at each sampling stage of the entire field application process. Additionally, theoretical models were built to represent the pressure and volume of hydrothermal fluid samples as a function of the preloading pressure of the precharged gas. Further, laboratory simulation and examination approaches were also adopted and compared, in order to obtain the volume change of the sample and accumulator chambers. By using theoretical models and the volume change of the two chambers, the optimized preloading pressure for the precharged gas was obtained. Under the optimized preloading pressure, the in situ pressure of the fluid samples could be maintained, and their volume was maximized. The optimized preloading pressure obtained in this study should also be applicable to other isobaric gas-tight hydrothermal samplers, by adopting a similar approach to pressure maintenance.