In this paper, the recombinant phage antibody techniques was used to construct, clone, screen, and express anti-rhEPO single chain antibody ScFv. The variable region genes of antibody were amplified by PCR from a hybridoma cell line D3 which secreted monoclonal antibody to rhEPO. The ScFv gene fragments were successfully cloned into phagemid vector pCANTAB5E. The recombinant phages were panned by rhEPO which was coated on a microtiter plate. After three rounds of panning, 8 clones were determined specifically binding to rhEPO antigen. The positive recombinant phagemids were extracted and transformed into non-suppressed E. coli HB2151. The soluble single chain antibody was expressed, and the specificity of the expressed ScFv was determined by ELISA. Western blot and Dot-blot. The result of SDS-PAGE indicated that the apparent molecular weight of the target peptide which is mainly in culture supernatant is 32 kD. The DNA sequence data showed that the ScFv gene included 783 bp, encoding 261 amino acids.