The binding constants of immobilized Cu (II) on PEG-IDA with imidazole and histidine at various solution pH values, salt conentrations and temperatures were determined by differential UV spectrophotometer. The bimodel binding behavior of basic solute was observed by the study of the salt effect. However, the formation of the coordinated compound is the dominated binding mechanism at the pH value higher than pKa of the deprotonation of imidazole nitrogen. There was no obvious effect of temperature on binding constant because of the complexity of binding mechanism. The binding behavior of several dipeptides and tripeptides with histidine at C- or N-terminal was also investigated and the results were explained by the “metal ion transfer” (MIT) hypothesis. Furthermore, the binding constants of synthetic heptapeptides with two histidine residues separated by different number of glycine residues were investigated to demonstrate the effect of histidine residues distance on the binding affinity. This study provides basic information of binding behavior of protein to immobilized metal ion.