In this study, p-tert-butyl-calix[4]arene derivatives selectively were synthesized. Then, new nanofibers were obtained from this p-tert-butyl-Calix [4]arene amid derivatives by the electrospinning method. In this study, an IMA nanofiber system via iminodiacetic acid as ligand and metals as Cu2+ for protein purification was developed. Copper (II) ions were chelated with IDA on nanofiber surfaces to prepare an immobilized metal affinity nanofiber (IMAN) for globulin depletion by the study of protein loading and release kinetics in various pH values. The loading and release of the globulin were monitored via fluorescence spectroscopy. The maximum amount of loaded protein onto 2.25 cm2 of the CAL-IDA (CIDA) nanofiber surface was found to be 0.092 μg in 6.0 of pH for 60 min. Any release from protein-loaded nanofibers did not perform in any pH values. Loading of the protein to nanofibers was also clarified by SEM, TEM, EDX, AFM, and FT-IR analysis.