Peptides, as the simplest biological recognition elements have been developed as probes to capture multitudinous targets. One important factor in fabricating peptide biochips is to immobilize peptides without losing their activity on a solid phase. To keep them functional, it is necessary to immobilize peptide in an oriented way. Calix[4]arene crown ether (CC) has been used as a protein linker molecule on the solid surface. In this study, calix[4]arene crown ether was self-assembled and modified on a gold surface. The calix[4]arene crown ether monolayer was characterized by EIS and SPR. An elaborately designed peptide probe Ac-CCPGCAAAARRRR-NH₂ is employed here for the assay of Zn²⁺. This tridecapeptide consists of a binding part (CCPGC), spacer (AAAA) and immobilization part (RRRR) to interact with a CC SAM. We compared the sensitivity and the specificity of the linker molecules with those of common attachment agents using a zinc ion binding peptide. The fabricated chip showed a superior sensitivity and a much lower detection limit than those chips prepared by other methods. Thus, the calix[4]arene crown ether chip can be used as a powerful peptide linker with a wide range of applications, including peptide–drug interaction, peptide–cell interaction, and an enzyme activity assay.