Gastrointestinal (GI) barrier dysfunction is an early pathogenic event in many complex diseases. Despite the routine applications of invasive tests, saccharide molecules are used noninvasively for assessing GI tract mucosal barrier function. However, currently available methods for quantification of saccharides molecules are costly and laborious. Simplified, reliable, and high-throughput methods are desired so that GI permeability testing can become routine and widely used. Here, we have developed a one-component system comprising of a naphthyl-pyridine core coupled to a boronic acid receptor, which can be used for early detection of saccharide biomarkers (i.e., lactulose) for applications related to GI barrier dysfunction. For quantitation of lactulose as a model biomarker, we have designed gold nanoparticle decorated surfaces in a highly scalable 96-well format to enable sensitive testing of lactulose within a broad range of concentrations. To tackle current challenges in saccharide biomarker sensing, we developed a hybrid sensing principle integrating two optical modalities (plasmonics and fluorescence) with a synthetic smart-probe (naphthyl-pyridinium) for monitoring GI permeability. This technology can be further developed as an affordable and portable diagnostic tool for GI permeability screening for routine use, facilitating early detection of various diseases affecting the GI tract.