The gold nanoparticles/colloidal carbon sphere hybrid material was used for the immobilization of protein and was developed in biosensing The hybrid material was fabricated by the assembly of gold nanoparticles onto the surface of colloidal carbon spheres which constructed a 3D antibody immobilization matrix on the glass carbon electrode and made the immobilized biomolecules hold high stability and bioactivity After the sandwich-type immunoreaction the formed HRP-labeled immunoconjugate showed good enzymatic activity for the oxidation of o-phenylenediamine by H2O2 The approach provided a linear response range between 5 and 250 ng/mL with a detection limit of 1 8 ng/mL The immunosensor showed good precision acceptable stability and reproducibility and could be used for the detection of human IgG in real samples which provided a potential alternative tool for the detection of protein in clinical laboratory (C) 2010 Elsevier Ltd All rights reserved