Tannins are chemically diverse polyphenols in plant-derived products that not only show diverse biological activities but also play a crucial role in determining the sensory attributes of food and beverages. Therefore, their accurate and cost-effective quantification is essential. Here, we identified a novel fluorescence quenching mechanism of different synthetic rhodamine fluorophores, with a high selectivity towards tannic acid (TA) and catechin-3-gallate (C3G) compared to a structurally diverse panel of tannins and polyphenols. Specific chemical conjugates of silicon-rhodamine with alkyl linkers attached to bulky apolar moieties had a limit of detection near 500 pM and a linear range spanning 5–100 nM for TA. We validated the assay on 18 distinct red wine samples, which showed high linearity (R2 = 0.92) with methylcellulose precipitation with no interference from anthocyanins. In conclusion, a novel assay was developed and validated that allows the sensitive and selective quantification of major astringency markers abundant in food and beverages.