Abstract: This study systematically evaluated the anesthetic efficacy and physiological effects of three anesthetics (eugenol, linalool, and MS-222) on juvenile orange-spotted grouper (Epinephelus coioides). Results showed a negative correlation between anesthetic concentration and induction time of anesthesia, while deep anesthesia prolonged recovery duration. Transportation experiments were conducted using concentrations that maintained the juveniles in a state of deep sedation (5 mg/L eugenol, 18 mg/L linalool, and 30 mg/L MS-222). After 18 h of anesthetized transportation, the survival rate of juveniles in the eugenol group was only 10.34% at 72 h post-recovery, whereas the survival rates in the other groups reached 100%. Physiological analysis revealed that MS-222 maintained stable energy metabolism by inhibiting muscle lactic acid accumulation and slowing glycogen consumption. Additionally, it reduced the activity of superoxide dismutase (SOD) in the liver, alleviated oxidative stress, and exerted minimal interference on the antioxidant system of the fish. None of the three anesthetics caused hepatic lipid peroxidation damage; however, eugenol and linalool were prone to inducing anaerobic metabolic stress in the fish, as indicated by a significant increase in muscle lactic acid content. The findings of this study provide a scientific reference for optimizing the anesthetized transportation of juvenile E. coioides and conducting subsequent related research.