Silkworms are useful bioreactors for heterologous protein expression when used in conjunction with the Bombyx mori nucleopolyhedrovirus(BmNPV) bacmid system. However, purification from silkworm hemolymph is difficult since it contains various kinds of proteins. In this study, we investigated an effective single-step method for the purification of affinity-tagged single-chain antibody variable region fragment (scFv) from silkworm larval hemolymph. A 5-fold higher expression level was obtained when scFv was fused with the His tag than when it was fused with the Strep II or GST tags. However, the His tag was inadequate for single-step purification since it led to the nonspecific binding of contaminants. The purification recoveries of GST-, Strep II-, and His-tagged scFvs were 91.8%, 43.7%, and 27.2%, respectively. The specific amount of single-step purified GST-tagged scFv was 2.2~2.7 fold higher than the amounts of the His- and Strep II-tagged constructs. The purities of Strep II- and GST-tagged scFvs in the eluent were 98.4% and 83.0%, respectively. Thus, both the short peptide Strep II and GST protein are suitable fusion tags for the affinity purification of proteins from silkworm larvae.