Copper nanoclusters (DPA@CuNCs) with red fluorescence were successfully synthesized by a one-step method based on D-penicillamine (DPA), which acted not only as a reducing agent but also as a stabilizer. The products were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, particle-size analysis, ultraviolet-visible spectrophotometry, and fluorescence spectrometry. When the excitation wavelength was 280 nm, DPA@CuNCs emitted bright red fluorescence at 640 nm with a fluorescence quantum yield of 5.8 %. Due to the inner filter effect, oxytetracycline (OTC) effectively quenched the fluorescence of DPA@CuNCs, and then DPA@CuNCs were applied to the trace detection of OTC. The method showed a good linear range for OTC from 5 to 60 & mu;mol/L, with a detection limit of 0.026 & mu;mol/L and a correlation coefficient R2 of 0.9983. Moreover, a paper-based sensor for the visual detection of OTC has been developed, which can conveniently and rapidly distinguish the concentration ranges of OTC through the color changes of the test papers.