In this study, a new red lysosome-targeted fluorescence off–on probe has been rationally designed, synthesized and applied for the highly selective and sensitive fluorescence sensing of carboxylesterase in live cells, sera and tissues. The probe was readily synthesized by coupling N-methylated benzoindole with 4-(acetyloxy)benzaldehyd, in which the acetyl group acted as a quenching and recognizing agent and the hemicyanine skeleton worked as the fluorescent signal unit. As a consequence, the prepared spectroscopic probe displays quite a low background signal. Upon addition of carboxylesterase, the latent probe could be easily hydrolyzed to liberate the fluorophore hydroxy hemicyanine and emit the off–on spectroscopic signal, which is desirable for obtaining sensitive detection with high specificity. The newly developed probe was successfully used to monitor and image carboxylesterase activity in complex biological samples such as serum, live cells and tissues. Moreover, this probe shows great promise for biomedical studies, including obesity disease, hepatocellular carcinoma and drug screening research.