Abstract: Ammonia nitrogen (NH₃-N) can serve as one of the key indicators of water quality. However, the NH₃-N level can rapidly increase over a short period in intensive aquaculture, due to farming density, feeding modes, and water renewal frequency. The high-level NH₃-N-induced stress can cause eutrophication damage to aquatic animals and even mortality. Therefore, it is crucial to detect the NH₃-N in water, in order to prevent water quality deterioration for the safety of aquatic life. Particularly, the current detection of NH₃-N is often complex, costly, time-consuming, and limited in detection range. This study aims to propose an accurate and rapid detection of high NH₃-N concentrations in water. A nitrogen-doped graphene quantum dots (N-GQDs) fluorescent probe was then constructed to detect the NH₃-N content in water. Firstly, the morphology and size distribution of the N-GQDs were examined using transmission electron microscopy (TEM). The lattice spacing of the N-GQDs was observed to be 0.20 nm, corresponding to the (100) lattice plane of the graphite structure. X-ray diffraction (XRD) analysis showed the diffraction peaks at 19° and 29° were attributed to the (101) and (002) crystal planes of graphene, respectively. Fourier transform infrared spectroscopy (FT-IR) analysis demonstrated that there was the presence of carboxyl groups (-COOH) on the surface of the N-GQDs. The N1s spectrum of X-ray photoelectron spectroscopy (XPS) indicated that the nitrogen was doped in the GQDs in the form of pyrrole nitrogen. The characterization results from TEM, FT-IR, XPS, and XRD confirmed that the N-GQDs with a graphene crystal structure were successfully synthesized. Additionally, the optical properties were performed on the N-GQDs fluorescent probe. Specifically, the N-GQDs exhibited an absorption peak at 320 nm in the UV-Vis spectrum, due to the π→π* transition within the C=C structure. Meanwhile, the fluorescence spectra showed that the optimum excitation and emission wavelengths of the N-GQDs were 350 and 450 nm, respectively. The aqueous solution of N-GQDs shared a pale yellow under daylight and then emitted blue fluorescence under a 360 nm UV lamp. The N-GQDs fluorescent probe showed a good linear response (R²=0.99) to NH₃-N concentration in the range of 0-9.0 mmol/L under the optimal experimental conditions (pH=7, N-GQDs concentration 0.8 mg/mL), with the detection limit concentration of 43.8 µmol/L and response time of 2 min. Furthermore, the stability, repeatability, and anti-interference properties of the fluorescent probe were carried out. The probe exhibited the excellent stability, and anti-interference properties against Na⁺, Hg⁺, Ag⁺, K⁺, Pb²⁺, Ca²⁺, Cd²⁺, and Cu²⁺. The fluorescence intensity of the probe still recovered to 77% of the initial value after three uses, indicating that the probe has good reproducibility. The response mechanism of the N-GQDs fluorescent probe to NH₃-N was analyzed by UV-Vis, FT-IR, and fluorescence lifetime decay analysis and Stern-Volmmer equation. The carboxyl groups on the surface of the N-GQDs exhibited electron-accepting properties. The pyrrole nitrogen doped into the GQDs carried a slight positive charge. Ammonia acted as a typical electron donor, due to the lone pair of electrons on the nitrogen atom. Therefore, it inferred that the photo-induced electron transfer occurred between ammonia and the N-GQDs, resulting in the dynamic quenching of N-GQDs fluorescence. Finally, the N-GQDs fluorescent probe was used to determine the NH₃-N in drinking, tap, and aquaculture water, with the recoveries of the spiked assay ranging from 75.03% to 128.16% and relative standard deviations (RSD) (n=3) lower than 13.53%. Consequently, the N-GQDs fluorescent probe also shared the potential applications in the detection of NH₃-N in water.