This study aimed to evaluate the effectiveness of eugenol for induction and anaesthesia recovery in hybrid Amazon catfish juveniles and to verify its effects after handling stress. Juveniles were exposed to different concentrations of eugenol: 0 (control), 10, 20, 30, 40, 50 and 60 μL L−1 (equivalent to 10.6, 21.2, 31.8, 42.4, 53.0 or 63.6 mg L−1). The fish were divided into two groups: anaesthetised with 53.0 mg L−1 eugenol and non-anaesthetised. Biochemical and haematological variables showed different responses for eugenol in handling (0, 1 and 5 h). Eugenol was recommended for sedation and anaesthesia with 21.2 and 53.0 mg L−1, respectively. In control fish, 25 and 50% mortality occurred at zero and one hour after handling stress. Plasma cortisol levels were higher in control fish than anaesthetised fish (0 h). Plasma AST and albumin levels were higher in control fish when compared to anaesthetised fish (0 and 1 h, respectively). In control fish, plasma cortisol, glucose, total protein and albumin levels were significantly lower at five hours after handling stress. Thrombocyte and neutrophil values were lower in control fish when compared to anaesthetised fish (1 and 5 h, respectively). The use of 53.0 mg L−1 eugenol is indicated as an anaesthetic for Amazon catfish and reduces post-handling stress by reducing mortality, improving biochemical responses and promoting increased immune system.