Abstract: To reveal the changes of biochemical characteristics( phenoloxidase,PO) of Penaeus vannamei in catalyze with different substrates. In this paper,L-dopa,methyldopa,catechol were taken as substrates,the optimum temperature of PO were measured by enzymatic oxidation with the three substrates,the p H optimum,enzyme kinetics constants K_m,V_max the value of activation energy were determined. The results showed that when PO enzyme catalyzed L-dopa,methyldopa and catechol,the optimum temperature were 45,40,30 ℃,the optimum p H was 6.8,7.2,8.0,the enzymatic kinetic constants were 2.5003,5.8661,1.8429 K_m mmol / L and V_max were 0.0624,0.1008,0.1692 ΔA / min,activation energy Ea were 15.2158,18.1981,10.4696 kJ / mol,respectively. Catechol binding with PO was stronger,but the combination was greatly influenced by the outside temperature,while the effects of temperature on PO combination with L-dopa and methyldopa were less.So it can be speculated that the simpler the substrate structure was the easier the combination with the active center of the PO; the stronger the enzyme substrate binding ability was the less activation energy was desired.In PO enzyme studies,if the temperature was included in factors,catechol should not be chosen as a substrate.