The objective of the study was to evaluate the efficacy of 2-phenoxyethanol (2-PE) as an anesthetic in great sturgeon under two experiments. First, fish were exposed to 0.1, 0.3, 0.5, 0.7 and 0.9 mL/L 2-PE, and time to induction (deep anesthesia) and recovery from anesthesia were measured. At concentration of 0.1 mL/L, 2-PE failed to induce deep anesthesia in fish, whereas at concentrations of 0.7 and 0.9 mL/L, all the fish were anaesthetized within 3 min of exposure. For assessing the impact of effective concentrations of 2-PE on physiological responses of great sturgeon, hematological indices, plasma metabolites, electrolytes, enzymes and cortisol levels were measured. The use of 2-PE induces a significant increase in RBC values at 0.3 mL/L concentration and a parallel increase in hemoglobin and hematocrit values. 2-PE anesthesia had no effect on WBC, MCV, MCH and MCHC levels when compared to control group. Serum glucose, cholesterol and cortisol levels were significantly high in 0.3 and 0.5 mL/L 2-PE. Moreover, AST levels were increased in fish exposed to the 0.3 mL/L 2-PE comparing with the control group (P < 0.05). There were no significant differences in serum levels of total protein, triglycerides, ALP, ALT, Cl−, Na+, K+, Ca2+ and Mg2+. In this study, alteration in hematological and serum biochemical indices was time-dependent. This study demonstrates that rapid induction of deep anesthesia with a relatively high concentration of 2-PE (0.7 and 0.9 mL/L) was associated with the lowest effects on the hematological and serum biochemical indices in great sturgeon and therefore would be recommended as eligible doses for hematological studies in this species.