Herein, a cost-effective, efficient, sensitive, and selective fluorescent sensor of graphene quantum dots doped with phosphorus and nitrogen (P, N-GQDs) is fabricated for direct determination of furazolidone in human urine and serum samples. The sensor is constructed via a facile hydrothermal method using urea and triphenylphosphine as nitrogen and phosphorus sources. The synthesized P, N-GQDs have a uniform size of less than 5 nm with high photoluminescence properties. The fluorescence intensity of P, N-GQDs at the wavelength of 447 nm is reduced in the presence of copper ions due to the electrostatic interaction between copper ions and the charged surface of the quantum dots. However, in the presence of furazolidone, the formation of a complex between the drug and the copper ions leads to the retrieval of the fluorescence emission of the P, N-GQDs, which is highly sensitive to furazolidone concentration. Under optimal conditions, the linear range of the calibration curve was 0.05-20 µmol L−1, and the detection limit was 0.009 µmol L−1, enabling P, N-GQDs as an effective and sensitive nanoprobe for furazolidone detection. The method was successfully applied to the direct measurement of furazolidone in real urine, serum, and tablet samples.