A novel thiosemicarbazide-based fluorescent sensor (AFC) was developed. It was successfully applied to detect hypochlorite (ClO⁻) with fluorescence quenching in bis-tris buffer. The limit of detection of AFC for ClO⁻ was analyzed to be 58.7 μM. Importantly, AFC could be employed as an efficient and practical fluorescent sensor for ClO⁻ in water sample and zebrafish. Moreover, AFC showed a marked selectivity to ClO⁻ over varied competitive analytes with reactive oxygen species. The detection process of AFC to ClO⁻ was illustrated by UV–visible and fluorescent spectroscopy and electrospray ionization–mass spectrometry (ESI–MS).