The adsorption mechanism of dipeptides namely Gly-Glu, Asp-Glu with protein layer was successfully examined on cytochrome c (Cyt.c) surface probing by fluorescent dye molecule. Molecularly thin Cyt.c layer was immobilized on poly(vinyl alcohol) (PVA) coated zirconium oxide (ZrO2) gel films by electrostatic interaction. Denaturation of Cyt.c was minimized on sol-gel derived ZrO2-gel film by PVA. The conformation of immobilized protein was verified by FTIR-ATR measurements. The fluorescent dye molecule was easily bound on the Cyt.c layer. The dye adsorbed protein thin film was soaked in two different dipeptides solution with various concentrations. The adsorption of dipeptides on Cyt.c thin film and consequent desorption of fluorescent dye from Cyt.c layer in dipeptide solutions was monitored by UV-vis absorption, fluorescence spectroscopy and quartz crystal microbalance method. The ion exchange mechanism on protein surface between fluorescent dye and dipeptides is thought to be responsible for dye desorption and formation of relatively strong electrostatic bonding between dipeptides with protein. The adsorption behavior curve of dipeptides was analyzed by Hill equation. The number of functional group together with size of dipeptides play an important role for different adsorption parameters obtained from this model.