Auto-phosphorylation of bacterial histidine kinases PhoR, PhoQ, and EnvZ has been investigated using adenosine-5'-[γ-ferrocene] triphosphate (Fc-ATP) as a cosubstrate for the first time. The study has been carried out in solution and on surface. Results from biochemical multiplex assay and surface electrochemical/optical methods are consistent, which successfully demonstrates that Fc-ATP is an efficient cosubstrate for histidine kinase auto-phosphorylations. The study also has discovered that the concentration of Fc-ATP influences the autophosphorylation efficiency. This developed methodology will provide a powerful tool in studying such biological processes towards further understanding of the involved mechanism.