Gold−silver alloy nanoclusters (AuAgNCs) were synthesized, protected by glutathione S-transferase (GST). The asprepared GST-AuAgNCs displayed strong (QY = 15.9%) fluorescence with two emission peaks around 477 and 653 nm under excitation of 395 nm. In this system, both the NC core and the organic shell contributed to the development of a "turn-on" ratiometric fluorescent probe for sensitively and selectively detecting oxytetracycline (OTC). First, the green fluorescence at 477 nm enhanced with the addition of OTC into GST-AuAgNC solution, whereas the red fluorescence at 653 nm remained unchanged. This "turn-on" ratiometric fluorescence may stem from the aggregation-induced emission effect and the reduction of Ag(I). The fluorescence ratio of GST-AuAgNCs displayed good linearity when the concentration of OTC was in the range of 0∼50 μM, and the detection limit was achieved down to 32.66 nM. Second, benefiting from the strong interaction between GST and OTC, the proposed method exhibited good anti-interference toward OTC in comparison with the common ions and natural amino acids. Moreover, the variation of ratiometric fluorescence intensity resulted in the corresponding red-to-green fluorescent color change, which could be employed to establish a simple paperbased analytical method for OTC colorimetric detection. Based on the superiority of high sensitivity and selectivity, as well as good accuracy and biocompatibility, the GST-AuAgNCs were successfully applied for detecting OTC in cells.