Protein phosphorylation is a wide existing protein post-translational modification in living organisms. This covalently linking mode between a certain amino acid and a phosphate group plays a sophisticated role in the regulation of protein stuctures and functions. Nowadays, nine amino acid residues have been found to have the capacity of phosphorylation, including histidine via a phosphamide bond. Although histidine phosphorylation has a significant regulatory function in both prokaryotic and eukaryotic cells, there has been seldom unimpeachable solution for the biologic research for a long time. Because of the diverse chemical properties of phosphohistidine per se from other phosphorylated amino acids, such as isomerism, chemical lability etc, it hydrolyzes easily during traditional handling methods. Due to the current progress in biochemistry and molecular biology, investigators have established innovative preparation, isolation and characterization strategies for phosphohistidine ad hoc. Therefore, the field begins to rapidly develop. Based on their chemical structures, this article analyzes the main physical and chemical properties as well as their reactivity of two phosphohistidine isomers, followed by the noval chemical biology tools and the main biological progresses.