Pearl gentian grouper (Epinephelus fuscoguttatus ♀×E. lanceolatus ♂) was studied to explore the cool acclimation process and evaluate its transport stress response through a series of biochemical indices. The effects of different cooling rates, water temperature, salinity, and fish-water ratios on the survival time of the pearl gentian grouper were determined. Response surface methodology was used to optimize the cool acclimation conditions, and a simulated transportation test was carried out on the pearl gentian grouper based on optimal conditions to determine the water quality indicators, serum biochemical indices, and liver antioxidant indicators at different time points; changes in the liver microstructures of the pearl gentian grouper were observed using an optical microscope. The optimal conditions for the acclimation process of pearl gentian grouper were obtained as follows: cooling rate of 1.2 ℃/h, temperature of 15.7 ℃, salinity of 24‰, and weight ratio of fish to water of 1∶ 5. Under these conditions the fish could survive for (55.6±1.7) h. In the simulated transportation, as the operation time increased, the total ammonia nitrogen concentration, nitrite concentration, and conductivity of the water increased rapidly. Transportation operation induced a gradual increase in serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities and a rapid increase in cortisol levels, and their content returned to normal after 48 h. Serum albumin, total protein, triglyceride and total cholesterol contents decreased significantly at 48 h, indicating that transportation stress led to the decomposition of proteins and lipids in the serum, which affected the composition and content of the serum. The activities of liver superoxide dismutase, hydrogen peroxidase, and glutathione peroxidase were elevated during transportation as a pathway to counteract the oxidative stress of the organism. However, with the prolongation of transportation time, the organism gradually depleted and reduced the production of these enzymes, and the enzyme activities showed reduced trends at 48 h. Optical microscopy revealed that adverse effects on the liver tissue of pearl gentian grouper gradually increased. After 24 h, the arrangement of liver cells became chaotic and the overall morphology of the liver was lost. The study showed that the optimized cool temperature acclimation conditions were suitable for the pearl gentian grouper in simulated transportation, and different stages of transportation exhibited varying changes in water quality indicators, serum biochemical indicators, liver antioxidant indicators, and microstructure. These results provide technical support for the preservation and transportation of pearl gentian grouper and other marine fish.