Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl -O-3,5,6-trichloro-2-pyridyl phosphorothioate) that is used in numerous agricultural and urban pest controls. The primary metabolite of chlorpyrifos is 3,5,6-trichloro pyridine-2-phenol (TCP). Because of its strong water solubility and mobility, this harmful metabolite exists in the environment in a large amount. Although TCP has potentially harmful effects on organisms in the environment, few studies have addressed TCP pollution. Therefore, this study was undertaken to investigate the effect of chlorpyrifos and TCP on the microsomal cytochrome P450 content in the liver, on the activity of NADPH-P450 reductase and antioxidative enzymes [catalase (CAT) and superoxide dismutase (SOD)], and on reactive oxygen species (ROS) generation and DNA damage in zebrafish. Male and female zebrafish were separated and exposed to a control solution and three concentrations of chlorpyrifos (0.01, 0.1, 1 mg L−1) and TCP (0.01, 0.1, 0.5 mg L−1), respectively, sampled after 5, 10, 15, 20 and 25 days. The results indicated that the P450 content and the NADPH-P450 reductase and antioxidative enzyme (CAT and SOD) activities could be induced by chlorpyrifos and TCP. DNA damage of zebrafish was enhanced with increasing chlorpyrifos and TCP concentrations. Meanwhile, chlorpyrifos and TCP induced a significant increase of ROS generation in the zebrafish hepatopancreas. In conclusion, this study proved that chlorpyrifos (0.01–1 mg L−1) and TCP (0.01–0.5 mg L−1) are both highly toxic to zebrafish.