Herbal medicines are widely used around the world, while some of them are associated with adverse effects like herb-induced liver injury due to oxidative/nitrosative stress resulted from hepatically-generated ROS/RNS. It is of significance to accurately evaluate herbal-medicine-induced hepatotoxicity, since it would help provide effective monitoring method of the safety of herbal remedies. Herein we designed a ratiometric nanoprobe for in vivo imaging hepatic injury induced by herbal medicine (polygonum multiflorum, PM) via specifically responding to NO generated in liver by PM, and with MSOT imaging the precise location of liver injury can be identified. The liposomal nanoprobe consists of a responsive dye (IX-2NH2) which could specifically respond to NO and the diketopyrrolopyrrole-based conjugated polymer (DPP-TT) as the internal reference. Thus we can realize ratiometric optoacoustic detection of herbal-medicine-induced liver injury with 3D information in mouse model in a noninvasive way.