Abstract: To assess the physiological and biochemical effect of Alizarin red S (ARS) on Spinibarbus sinensis juveniles, antioxidant enzymes (SOD, CAT, GSH-Px) activity and malondialdehyde (MDA) content of liver, brain and gill tissue were detected after 24h treatment in ARS. The results showed that brain GSH-Px activity increased continuously with rising concentration of ARS, other antioxidant enzymes activities in brain and antioxidant enzymes activities in both gill and liver were increased firstly and then decreased, which showed a parabolic dose-response relationship with the ARS concentration. Antioxidant enzyme activity in liver, gills and brain reached to the maximum at the concentration of 300, 300 and 400 mg/L, respectively. The MDA content of brain and liver firstly decreased and then increased with the rising concentration of ARS, but gill MDA content was increased continuously. These results suggest 120-200 mg/L of ARS for marking S. sinensis juveniles.