The quantitative profiling of residue reactivity in proteins promotes the discovery of covalent druggable targets for precise therapy. Histidine (His) residues, accounting for more than 20% of the active sites in enzymes, have not been systematically characterized for their reactivity, due to lack of labeling probes. Herein, we report a chemical proteomics platform for the site-specific quantitative analysis of His reactivity by combination of acrolein (ACR) labeling and reversible hydrazine chemistry enrichment. Based on this platform, in-depth characterization of His residues was conducted for the human proteome, in which the rich content of His residues (>8200) was quantified, including 317 His hyper-reactive residues. Intriguingly, it was observed that the hyper-reactive residues were less likely to be the sites for phosphorylation, and the possible mechanism of this antagonistic effect still needs to be evaluated in further research. Based on the first comprehensive map of His residue reactivity, many more residues could be adopted as the bindable sites to disrupt the activities of a diverse number of proteins; meanwhile, ACR derivatives could also be used as a novel reactive warhead in the development of covalent inhibitors.