Abstract <p>As an antibiotic, tetracycline (<b>TC</b>) is widely used and plays an important role in anti-infection treatment, so the quantitative detection of TC is of great significance. The purpose of this research is to develop a novel synthesis of silver nanoclusters stabilized with non-thiol small molecules and their application in the detection of tetracycline. Silver nanoclusters (<b>AgNC</b>s) were synthesized under optimized conditions using Acid Red 94 as a stabilizer, silver nitrate solution as the raw material, and sodium borohydride as the reducing agent. The optimal excitation wavelength of the synthesized AgNCs was 330 nm, and the optimal emission wavelength was 435 nm. It was found that TC could significantly quench the fluorescence of the synthesized AgNCs. The quenching value (Δ<i>F</i>) of the fluorescence intensity of the AgNCs has a good linear relationship with the TC concentration in the range of 10.0–50.0 μmol/L, and the detection limit was 2.3 μmol/L. Therefore, a method for TC detection was established, which was further verified by the spiked recovery method in water samples. The spiked recovery rate ranged from 101.7 to 103.8%, indicating that the established method has potential application value for the quantitative detection of TC in water samples. Additionally, a visual quantitative detection method for TC was achieved based on smartphone and colorimetric analysis software using AgNCs as fluorescent probes.</p>