Abstract: Water flow is one of the important ecological factors that affect the physiology and growth of fish. Studying the stress level changes of Plectropomus leopardus under flow velocity stress, and clarifying the maximum flow velocity that the fish can tolerate, can provide a theoretical basis for the cage culture site selection, intensive flow culture and deep-sea aquaculture working vessel. Taking P. leopardus Body length (11.38±1.48) cm, body mass (34.71±11.57) g as the research object, we designed a hydrostatic control group (0 cm·s⁻¹ or 0 BL·s⁻¹, body length·s⁻¹) and three experimental groups (11.4, 22.8 and 34.2 cm·s⁻¹ corresponding to 1, 2 and 3 BL·s⁻¹) by a self-made fish swimming experimental device, so as to explore the effect of 40-min water flow stimulation on its tail beat frequency change, blood glucose, lactic acid and cortisol content in blood. The results show that there was a linear correlation between the tail beat frequency and water velocity. The blood glucose and cortisol levels increased linearly but the lactic acid increased non-linearly with increasing tail beat times. The flow rate over 2 BL·s⁻¹ resulted in a significant increase in the cortisol in P. leopardus blood (P<0.05). When the flow rate reached 3 BL·s⁻¹, lactic acid and blood glucose levels increased significantly (P<0.05). In conclusion, the upper limit of tolerance to flow velocity of P. leopardus was 2 BL·s⁻¹. When the flow velocity of aquaculture water was higher than 2 BL·s⁻¹, the stress level and metabolic load increased significantly and the stress effect appeared (P<0.05). The results provide references for the aquaculture working vessel sloshing suppression and the site selection of cage aquaculture waters.