Hydrophobins are small fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. A new hydrophobin gene hgfI was identified recently from the edible mushroom Grifola frondosa. In this paper, the cloning, expression, purification, and polyclonal antibody preparation of the HGFI were described. The hgfI gene was cloned into pET-28a expression plasmid at the EcoRI and NdeI restriction sites and then transformed into Escherichia coli BL21 strain. SDSPAGE analysis showed that recombinant HGFI protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. The expressed recombinant HGFI protein was purified by electroelution because its inclusion body was insoluble in traditional processing method. After a desalting procedure with Sephadex G-25, the recombinant HGFI protein was used to immunize adult rabbits following standard protocol. ELISA and western blot analysis indicated that the produced antiserum could detect both HGFI protein expressed in the prokaryotic (E. coli) and in the eukaryotic cells (G. frondosa). Furthermore, the antiserum was used to determine the localization of HGFI protein in G. frondosa cells using an immunofluorescence technique. The results demonstrated that HGFI protein was localized in the cell wall, especially at the budding position of hypha. The polyclonal antibody against HGFI will facilitate further production and functional study of HGFI protein.