Water environment pollution caused by the widespread application of beta-cypermethrin (BCP) and chlorpyrifos (CPF) in agriculture has attracted extensive concern of the world. In this study, zebrafish was used as a model to investigate the individual and joint toxicity of BCP and CPF. In the acute toxicity test, 3 hpf embryos were exposed to various concentrations of CPF, BCP and their binary mixtures (MIX) for 96h. The results indicated that these two pesticides and mixtures induced malformation and death in larvae, and affected hatchability. These two pesticides in mixtures were verified to act together in a synergistic manner under experimental conditions. Oxidative stress assaying manifested that CPF, BCP and MIX altered CAT, SOD and GST activities and MDA content, resulting in oxidative damage in larvae. By pathology analysis, CPF (236μg/L), BCP (5.9μg/L) and MIX (236μg/L CPF+5.9μg/L BCP) were found to trigger liver lesions and promote apoptosis in tissues. The transcriptome sequencing suggested that ECM- receptor interaction, focal adhesion, cell cycle, DNA replication, phototransduction and adherens junction pathways were closely associated with the toxicity of these two pesticides.