In this study, the optimization of fabrication conditions was accomplished to make immuno-strip biosensor by the combination of enzyme linked immunosorbent assay (ELISA) and immuno-chromatographic strip techniques for the detection of Escherichia coli O157 : H7. Optimal fabrication conditions of capture antibody concentration, detection antibody concentration, and additive composition of running buffer solution were determined. Optimal concentration was determined as 1.0 mg/mL for both of capture antibody and detection antibody. A composition of 0.5% Tween20 and 3% BSA were selected as optimal additive for buffer solution to prevent non-specific binding.