Hydrazine (N2H4) produced by industries is distributed into different environments, and seriously threatens ecology and human security. Hence, it is important to develop probes that detect N2H4 in various environments. In this study, a novel N2H4 fluorescent probe was prepared based on a dibenzothiazole derivative (DBTD). The obtained DBTD probe demonstrated a strong ratio of colorimetric detection of N2H4, a rapid response, and good selectivity and sensitivity (detection limit 0.438 μM). Based on its good performance, the DBTD probe was successfully applied for the determination of trace N2H4 in water, cells, and zebrafish. In addition, the results of the fluorescence colocalization experiment demonstrated the lysosomal-targetable ability of DBTD.