It is well accepted that peroxynitrite (ONOO⁻) plays a crucial role in various physiological and pathological processes. Thus, the detection and imaging of ONOO⁻ in vitro and in vivo with high selectivity and sensitivity is of great significance. Here we report two simple benzothiazole-based fluorescent chemosensors, BS1 and BS2. Under physiological pH, both probes could quickly sense ONOO⁻ with a remarkable “turn-on” fluorescence signal at 430 nm. The limit of detection (LOD) of BS1 and BS2 toward ONOO⁻ was 12.8 nM and 25.2 nM, respectively, much lower than the reported values. Experimental results indicated that BS1 with a diphenyl phosphonate unit presented higher selectivity for ONOO⁻ than BS2. Furthermore, based on the advantages of lower cytotoxicity and pH-stabilities of BS1, probe BS1 was successfully employed to detect and image ONOO⁻ in HepG2 cells. More importantly, we used BS1 to successfully showcase drug-induced hepatotoxicity via imaging ONOO⁻ upregulated by acetaminophen (APAP), and also evaluated the remediation effect of GSH. All the results illustrated that the fluorescent probe BS1 has great potential for the detection of ONOO⁻ and to further uncover the roles of ONOO⁻ during the drug-induced liver injury (DILI) process.