Herbal medicines and dietary supplements are widely used worldwide. However, reports have revealed that a large number of herbal medicines are implicated in the hepatic damage, which is referred to as Herbal medicines-induced liver injury (HILI). Traditional diagnostic methods for HILI include histological and serological assessments, which fail to offer real-time and unambiguous visualization of hepatotoxicity in vivo. Herein, we constructed a second near-infrared (NIR-II) luminescent nanoprobes to achieve accurate, high-efficiency, and non-invasive detection of HILI by the assembly of lanthanide-based NIR-II luminescence nanoparticles with a chromophore with absorption band in the NIR-II region. Specifically, the NIR-II luminescence of the lanthanide nanoparticles can be effectively quenched by the chromophores on the surface of nanoparticles via an absorption competition-induced quenching effect, and subsequently recovered by the pathophysiological biomarker peroxynitrite (ONOO−) generated in early HILI. It is demonstrated that the nanoprobes are capable for the screening of liver toxins in a noninvasive pattern, which is more convenient than the conventional diagnostic approaches. Finally, our luminescence detection approach offers an immediate testing technique for the identification of hepatotoxic components of herbal medicine such as colchicine, monocrotaline and stibene glucoside, providing a high-throughput screening strategy for herbal medicine-induced hepatotoxicity.