A carbon nanofiber-based luminol-biotin probe was synthesized for the sensitive chemiluminescence (CL) detection of a target protein by grafting luminol and biotin onto an oxidized carbon nanofiber. This carbon nanofiber was prepared by chemical vapor-deposition with methane in the presence of the Ni-Cu-MgO catalyst, which was followed by oxidization with HNO3-H2SO4 to produce a carboxyl group on the surface of the nanofiber. The material was grafted with luminol and biotin by means of a standard carbodiimide activation of COOH groups to produce corresponding amides. The substance was water-soluble and thus could be utilized as a sensitive CL probe for a protein assay. The probe showed highly specific affinity towards the biotin-labeled antibody via a streptavidin-biotin interaction. The detection limit for this model assay was approximately 0.2 pmol of the biotinized IgG spotted on a polyvinylidene fluoride (PVDF) membrane. Nonspecific binding to other proteins was not observed. Therefore, the synthesized carbon nanofiber-based CL probe may be useful for a sensitive and specific analysis of the target protein.