A method based on spectroelectrochemistry to monitor intramolecular electron transfer in [Ni-Fe]-hydrogenase was developed. The electron transfer generated a color change in manganese-tetra(4-pyridyl)porphyrin (MnTPyP), and time-resolved measurement of the spectral change allowed determination of the intramolecular electron transfer rate. A MnTPyP-hydrogenase-electrode was prepared as a model system to monitor an intramolecular electron transfer, and spectroelectrochemical measurement was carried out. An intramolecular electron transfer of [Ni-Fe]-hydrogenase, which is a rate between distal [4Fe-4S] and [Ni-Fe], was measured by the developed system.