Lateral flow assay (LFA) type of biosensors have been popular due to cost-effectiveness and easy-interpretation for instant results, most common examples of applications being pregnancy tests, food safety or medical diagnostics. There are several examples of reports with high sensitivity, including pre-concentration of the sample by magnetic pull-down. However, sensitivity and direct detection designs with aptamers has been a limiting factor for developing aptamers-based LFA assays. In this study, we report a lateral flow design based on aptamer-gated silica nanoparticles to develop high sensitivity and direct bacterial assay by shifting aptamers-target interaction to conjugation pad. Aptamer-gated silica nanoparticles-based biosensors were reported for their high sensitivity, specificity and label-free detection for small molecules and whole cells. This label-free strategy for LFA can determine L. monocytogenes in minced chicken matrix at less than 5 min with a limit of detection (LOD) of 53 cells in one mL samples.