Background: Metronidazole is widely used due to its clinical excellence in treating systemic or local infections caused by anaerobic bacteria. However, it is easily soluble in water, not easy to biodegrade and adsorb and stays for a long time in environments, causing great harm to human health and food safety. Therefore, it is important to choose highly selective and sensitive methods for metronidazole content determination in environments. In this paper, the edible fungus Boletus speciosus was used as the carbon precursor to successfully prepare carbon dots by one-step hydrothermal method, and were used to analyze metronidazole. Methods: Characterization of the prepared carbon dots from B. speciosus (Bs-CDs) were studied by Transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and X-Ray Diffraction. Results: The linear equation was y=0.06231+0.01099x (R2=0.9970) with a metronidazole concentration of 2.5~50 μM, and the detection limit was 71 nM. The fluorescence quenching mechanism of Bs-CDs detecting metronidazole belonged to the internal filtration effect. Bs-CDs were applied to detect metronidazole in actual water samples, presenting good sensitivity and a high recovery rate (97.0~106.0%). Conclusion: It provides a new idea for applying carbon dots in metronidazole content detection.