Antibodies can distinguish not only differences in amino acid sequences (primary structure), but also differences in three-dimensional structure and thus may be useful for detecting the conversion of prion proteins, especially in vivo. For diagnosis, we prepared chicken single chain variable fragment (scFv) antibodies that specifically recognized a prion protein using a phage display approach. As antigen, mouse prion protein (MoPrP) 138-153 containing YYR residues was conjugated with KLH. Total RNA was extracted from the splenocytes of an immunized chicken, and the cDNA of scFv was ligated in a phagemid vector. The phage display scFv library was panned against the peptide antigen four times. Twenty-three scFv phage clones that tested positive using ELISA with the peptide antigen were then reacted with recombinant mouse prion protein (23-231), mouse brain homogenate, mouse neuroblastoma Neuro-2a, recombinant human V129 and M129 prion proteins, and human glyoma T98G using ELISA, immunoblotting analysis, and immunocytochemistry. The results suggested that the scFv phage clones were useful for detecting mouse and human prion proteins.