The present study aimed to determine the optimal concentration of three anaesthetics, name MS222, ketamine, and tobacco extract, in grass carp juveniles (107. 78± 22. 11 g body weight and 226. 74± 13. 06 mm total body length). Treatments were as follows: 100 mg L-1 of MS222 (control group), 5. 5, 8, 11, 18. 5, and 22 mg L-1 of ketamine, and 10, 13. 33, 20, 26. 27, and 40 mg L-1 of tobacco extract. In addition, changes in blood and biochemical parameters were measured at different concentrations. Considering the anaesthesia induction and recovery durations times, the optimal concentration for anaesthesia of studied substances was obtained as follows: 100 mg L-1 of MS222 (139. 00± 20. 00 seconds), 18. 5 mg L-1 of ketamine (140. 10± 20. 20 seconds), and 40 mg L-1 of tobacco extract (174. 00± 20. 09 seconds). Based on the results, the shortest recovery time from anaesthesia was observed at 26. 27 mg L-1 of tobacco extract (870. 00± 5. 3 seconds) and 8 mg L-1 of ketamine (967. 00± 5. 5 seconds), respectively. The results indicated that the anaesthesia induction and recovery duration was dependent on the concentration of anaesthetics. Moreover, significant differences were observed between the three anaesthetics in terms of blood parameters. The study findings also revealed a significant difference between treatments in biochemical parameters (cortisol, glucose, and lactate).