Nitrogen-doped carbon quantum dots (N-CQDs) with strong fluorescence were prepared by a one-step hydrothermal method using natural biomass waste. Two efficient fluorescent probes were constructed for selective and sensitive detection of oxytetracycline (OTC). The synthesized N-CQDs were characterized by UV-visible absorption spectra, fluorescence spectra, Fourier transform infrared spectroscopy (FT-IR), X-ray photon spectroscopy (XPS), atomic force microscopy (AFM), and high-resolution transmission electron microscopy (HRTEM), which proved that the synthesized N-CQDs surface were functionalized and had stable fluorescence performance. The basis of N-CQDs detection of OTC was discussed, and various reaction conditions were studied. Under optimized conditions, orange peel carbon quantum dots (ON-CQDs) and watermelon peel carbon quantum dots (WN-CQDs) have a good linear relationship with OTC concentrations in the range of 2–100 µmol L⁻¹ and 0.25–100 µmol L⁻¹, respectively. ON-CQDs and WN-CQDs were both successfully applied in detecting the OTC in pretreated tap water, lake water, and soil, with the recovery rate at 91.724–103.206%, and the relative standard deviation was less than 5.35%. The results showed that the proposed N-CQDs proved to be green and simple, greatly reducing the detection time for OTC in the determination environment.