Herein, a novel bright-green and highly fluorescent polyethyleneimine capped copper nanoclusters (CuNCs@PEI) has been hydrothermally synthesized, which is employed as a fluorescence probe to determine rifampicin. Under optimum synthetic conditions, the morphology, surface chemistry and optical features of resulting CuNCs@PEI are well characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, UV-Vis absorption spectra, transient and steady-state fluorescence. In comparison to previous reports, the new species with a larger diameter of approximately 3 nm exhibit higher fluorescence quantum yield (QY) up to nearly 10.7%, and demonstrate tolerability to extreme pH, high ionic strengths as well as long-term UV irradiation. These excellent optical properties ascribe to unique grain size and surface chemical features of products, which are further discussed in this work. In addition, we employed as-prepared CuNCs@PEI to detect rifampicin based on fluorescence quenching phenomenon. The fluorescence intensity of probe is linearly proportional to rifampicin over the concentration rage from 0 to 20 μM, where the limit of detection (LOD) is 50 nM. Besides, this probe can be also used to assay rifampicin in serum samples with satisfactory recoveries.