A highly selective method has been developed for the fluorometric determination of chlortetracycline (CTC) among other tetracycline antibiotics (TCs). It is making use of fluorescent Au/Pt nanoclusters (NCs) capped with polyethyleneimine (Au/PtNCs@PEI). The nanoprobe, with a green emission peaking at 512 nm, was synthesized by an environmentally friendly hydrothermal method. The capped NCs have a large Stokes shift (∼150 nm), are insensitive to extreme pH values and high ionic strength, and are excellently photostable under UV irradiation. In the presence of CTC, the fluorescence of the capped NCs is quenched due to aggregation. The effect is also found for tetracycline, oxytetracycline and doxycycline. This shows that sensitive but non-selective detection of such TCs is possible. However, CTC is specifically complexed by Al(III) ions, and this generates a strong fluorescence peaking at 520 nm even though the fluorescence of the capped NCs is fully quenched. Obviously, the effects are caused by CTC only, and this enables CTC to be specifically recognized by an "on-off-on" strategy. Fluorescence increases linearly in the 0.5 to 10 μM CTC concentration range, and the limit of detection is 0.35 μM. The method was successfully applied to the determination of CTC in (spiked) milk, and the recoveries suggest that this fluorescent probe is an effective tool for detecting CTC in foodstuff. Graphical abstract Schematic illustration and photographic images of the luminescence quenching response of Au/Pt nanoclusters (Au/PtNCs) toward chlortetracycline (CTC) (from on to off), and then the recovery upon Al addition (from off to on).