This study aims to produce immunotherapeutic human monoclonal antibodies in the form of single chain antibodies (ScFv) against staphylococcal enterotoxin A (SEA) by using phage display human antibody library. Recombinant SEA protein (rSEA) was produced by amplified the sea full length from Staphylococcus aureus strain ATCC 13565 and subjected to host protein expression system. The purified rSEA was used in a “single-round” phage biopanning against an established phage display human antibody library and the rSEA specific phage population was obtained. Twenty-eight individual phage clones with high binding affinity ScFv to the rSEA were obtained after a phage ELISA using the rSEA as an antigen to coat a microtiter ELISA plate and the bovine serum albumin as a negative antigen control. Individual phages were used to transfect non-suppressor E. coli bacteria for production of the human soluble ScFv. Two transformants carrying scFv-phagemids were obtained. The scFv derived from various E. coli transformants revealed high DNA banding pattern diversity (different RFLP) implying either epitope or affinity diversity of the expressed ScFv. Expressed ScFv was purified from individual E. coli transformants by using an anti-E-tag column. The efficiency of the ScFv in neutralizing the rSEA was investigated by inhibition of lymphocyte cells proliferation assay. The results showed one selected ScFv from phage clone is successes to inhibit the lymphocyte proliferation by rSEA. This study successfully produced human antibodies ScFv that can inhibit rSEA acitivity. These antibodies may be use for treatment in patient who got staphylococcal enterotoxin.