Abstract: In order to investigate the effects of different stress temperatures and stress durations on the activities of antioxidant enzymes (SOD, CAT, GPX) and immune enzymes (ACP, AKP, LYS) in the coelomic fluid of Holothuria leucospilota, this study employed response surface methodology (RSM) to evaluate the combined effects of temperature (10  ℃-38  ℃) and time (1 d-5 d) on the activities of these enzymes. Multi-response optimization was further applied to determine the maximum response conditions of coelomic fluid enzyme activities to temperature-time stress. The results showed that when the temperature deviated from the optimum (25  ℃), the activities of SOD, GPX and ACP in the coelomic fluid of H. leucospilota increased significantly, while CAT and AKP exhibited different response patterns. The first-order and second-order effects of both temperature and time significantly influenced the activities of SOD, GPX, ACP and AKP (P < 0.05), with temperature showing a stronger effect. The interaction between temperature and time significantly affected SOD, AKP and ACP (P < 0.05), whereas the interactive effects on GPX and LYS activities did not reach a significant level (P > 0.05). The coefficients of determination (R²) of the quadratic polynomial regression models ranged from 0.90 to 0.98, indicating good fit. Model optimization and validation experiments showed that the maximum response condition of coelomic fluid enzyme activities to temperature-time stress in H. leucospilota was at 10.00 ℃ and 2.47 d, with a desirability of 0.864, and the average agreement rate between measured and predicted values of each enzyme was higher than 89.61%. The changes in coelomic fluid enzyme activities under temperature-time stress could be well fitted by quadratic regression curves. The results reveal the specific temporal response strategies of the antioxidant and immune enzyme systems to temperature stress, and provide a scientific basis for temperature regulation and management in H. leucospilota aquaculture.