Lateral flow assay (LFA), or the immunochromatographic strip test, is popular to use for rapid and sensitive immunoassays. Gold nanoparticles (GNPs), due to tunable optical characteristics and easy manipulation of size or shape, represent an attractive approach for LFA technology. Since most enterohemorrhagic infections result from water and food contaminations of Escherichia coli O157:H7, selective and rapid detection of this organism in environmental and biological complexes is necessary. In this study, optimized parameters of antibody (Ab)-based LFA for rapid detection of pathogenic E. coli O157:H7 are described. GNPs were used as visualizing agents. The measuring parameters include the Ab concentration on the capture lines, the concentration of gold conjugate, and flow rate. M180 and 36 nm were the ideal membrane and GNP size, respectively, for bacterial detection of LFA. The target, E. coli O157:H7, could be detected with a visual limit of detection of 105 cfu/mL in 3-5 min. Selectivity of the system was very high and the target was recognized by developed strips, regardless of its presence singly or in mixed bacterial samples.