A sensitive fluorescent sensor has been developed for the determination of tetracycline (TC) using adenine thymine (AT)-rich single-stranded DNA (ssDNA) templated copper nanoclusters (CuNCs) as a fluorescent probe. Fluorescent ssDNA-CuNCs were synthesized by employing AT-rich ssDNA as templates and ascorbic acid as reducing agents through a facile one-step method. The as-prepared ssDNA-CuNCs exhibited strong fluorescence with a large Stokes shift (240 nm) and stable fluorescence emission. In the presence of TC, the fluorescent intensity of ssDNA-CuNCs was obviously decreased through the inner filter effect, due to the spectral overlapping between ssDNA-CuNCs and TC. Under the optimal conditions, the strategy exhibited sensitive detection of TC with a linear range from 2 nM to 30 μM and with a limit of detection of 0.5 nM. Furthermore, the sensor was successfully applied for the detection of TC in milk samples. Therefore, it provided a simple, rapid, and label-free fluorescent method for TC detection. Graphical Abstract This is a visual representation of the abstract. [ABSTRACT FROM AUTHOR]