To immobilize cytochrome c (cyt. c) on ITO electrode with keeping its direct electron transfer (DET) activity, 10-carboxydecylphosphonic acid (10-CDPA) self-assembled monolayer (SAM) film was formed on ITO electrode. After 100 times washing process with exchanging phosphate buffer saline solution in the cell to fresh one, in situ slab optical waveguide (SOWG) absorption spectral measurement proved that about 80% of cyt. c immobilized on 10-CDPA modified ITO electrode was adsorbed on ITO electrode. Additionally SOWG spectral change of cyt. c between oxidized and reduced forms was observed with setting the ITO electrode potential at 0.3 and -0.3 V vs. Ag/AgCl, respectively showing DET reaction between cyt. c and ITO electrode occurred. About 30% of a monolayer coverage was estimated from the coulomb amount in the surface area of oxidation and reduction peaks on cyclic voltammetry (CV) data. CV peak current maintained 84% for ITO electrode modified with 10-CDPA SAM film after 60 min continuous scan with 0.1 V/sec from 0.3 and -0.3 V vs. Ag/AgCl.