Phage display technology has been widely used to isolate antibodies with specific properties. The objective of this study was to isolate anti-rhTNF-alpha scFvs from phage display library. However, the inserted genes of eluted phages were either incorrect or truncated. In order to address this issue, bioinformatics was applied to facilitate the screening of the eluted phages. The alignment of the sequencing results was performed with the software ClustalW. The gene of scFv (F6) was assembled by ligating together the identical VH and VL fragments and then analyzed by using program BLASTX. F6 was identified to share 80% sequence identity with a human anti-TNF-alpha scFv. Subsequently, the conformation of F6 binding to hTNF-alpha predicted by docking assay showed that F6 could bind to hTNF-alpha via the six CDRs. Finally, ELISA assay and Western blot analysis indicated that F6 might bind to rhTNF-alpha specifically. Biological assay demonstrated that F6 might neutralize rhTNF-alpha-induced cytotoxicity in L929 cells. In conclusion, F6 could be a candidate for further investigation, based on the experimental data and the prediction by bioinformatics.