The purpose of this study was to investigate the detoxification effect of extracts from the petal of Crocus sativus L. (PCSE) on acetaminophen (APAP) induced liver injury in mice and its related mechanisms. LC-MS/MS analysis was used to identify the main components in PCSE, and an APAP-induced acute liver injury model in mice was constructed to evaluate the detoxification effect of PCSE. Liver tissue H&E staining, liver function indexes including ALT and AST, pro-inflammatory cytokine including TNF-α and IL-6, as well as hepatic tissue oxidative stress levels were examined. In addition, in vitro APAP induced cell was also prepared, apoptosis levels were detected by AO/EB staining, ROS fluorescence intensity was analyzed as well as the expression levels of apoptosis-related proteins and Nrf2/HO-1 pathway-related proteins were detected by western blot, to investigate the mechanism of PCSE's action in ameliorating liver injury. The results showed that PCSE can improve the survival rate of APAP induced mice, decrease ALT, AST, TNF-α and IL-6 levels, and ameliorate the liver injury induced by APAP. Furthermore, the mechanism research suggested PCSE attenuated oxidative stress and apoptosis in APAP-induced liver cells, as well as activated the Nrf2/HO-1 signaling. In summary, PCSE possesses potential detoxification effects on APAP induced liver injury by inhibiting hepatocyte apoptosis via regulating Nrf2/HO-1 signaling, which provides more possibilities for the drug selection for the treatment of liver injury in clinical practice.