Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposure to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120 h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120 h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches, such as ABPP, can, therefore, be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology.