The α-chain of FcεRI (FcεRIα) plays a critical role in the binding of IgE to FcεRI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human FcεRIα using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcRε27, FcRε51, and FcRε70) through two rounds of biopanning selection. The purified soluble scFv, FcRε51, inhibited the binding of IgE to recombinant FcεRIα, although both FcRε27 and FcRε70 showed fine binding specificity to FcεRIα. Since FcRε51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcRε51 to FcεRIα was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to FcεRIα (K_d = 0.5 nM). With these characteristics, FcRε51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.