we developed a novel H3g27Cr peptide probe mimicking Histone lysine crotonylation, which could more effectively recognize Histone Kcr-interacting proteins through covalent proximity-induced bioconjugation. We initially validated the orthogonality and cysteine reactivity of crotonyl-like group through protein-ligand binding models of PDZΔRGS3 and PLCγ1-c SH2, and further showed the feasibility of H3g27Cr to covalently bind the known Kcr “eraser” HDAC1. With the H3g27Cr probe in hands, we successfully identified and validated one unknown H3K27Cr-interacting protein, the transcriptional activator STAT3, from AP-MS experiment in A549 cell lysates. The candidate protein STAT3 was found that directly bound to the Histone H3K27Ac/Cr in XL-MS and pull-down assays, and this interaction might reduce the H3K27Ac/Cr levels.