In this study, the silver nanoclusters (Ag NCs) with aggregation-induced emission property were synthesized successfully by reflux method using silver nitrate (AgNO3) as raw material and N-acetyl-L-cysteine (NAC) as the reductant and capped ligand. The synthesis process was simple, environmentally friendly and the process did not require the addition of other surfactants or common reductant such as sodium borohydride, ascorbic acid. The obtained Ag NCs possessed the special Ag(0)@Ag(I)–SR core-shell structure with aggregation-induced emission characteristic. The Ag NCs exhibited strong green luminescence with an optimal emission wavelength of 494 nm, high quantum yield of 0.44, microsecond long life of 11.07 μs and high content of Ag(I) component. In addition, the obtained Ag NCs were used to detect the tetracycline (TC) with the range of linearity from 1.12 μM to 230 μM and a detection limit of 0.47 μM on account of the quenching mechanism of the internal filtration effect (IFE) and the static quenching effect between tetracycline and Ag NCs. The proposed sensing method based on NAC@Ag NCs showed good sensitivity and selectivity for tetracycline, wide range of linearity, low detection limit and strong anti-interference ability. This method had been applied to detect tetracycline concentration reliably and accurately in actual sample milk.