Abstract: Ammonia nitrogen (ammonia-N) serves as a key indicator of water quality. The high level ammonia-N can lead to eutrophication and a decline in water quality. And due to its toxicity, ammonia-N is highly harmful to aquatic animals. In intensive aquaculture, ammonia-N levels can rapidly increase over a short period due to factors such as farming density, feeding methods, and water renewal frequency. This increase can cause ammonia-N-induced stress and damage in aquatic animals and even mortality. Therefore, detecting ammonia-N in water is crucial for preventing water quality deterioration and ensuring the safety of aquatic life. However, current methods for detecting ammonia-N are often complex, costly, time-consuming, and with a narrow detection range, making it challenging to achieve rapid detection of high ammonia-N concentrations in water. In this study, a nitrogen-doped graphene quantum dots (N-GQDs) fluorescent probe was constructed for the detection of ammonia-N content in water. First, a microscopic characterization analysis of the N-GQDs was conducted. Transmission electron microscopy (TEM) was used to examine the morphology and size distribution of the N-GQDs. 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 diffraction peaks at 19° and 29°, attributed to the (101) and (002) crystal planes of graphene, respectively. Fourier transform infrared spectroscopy (FT-IR) analysis demonstrated the presence of hydroxyl groups (-OH) and 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 N-GQDs with a graphene crystal structure were successfully synthesized in this study. Additionally, the optical properties of the N-GQDs fluorescent probe were analyzed. Due to the π→π* transition within the C=C structure, the N-GQDs exhibited an absorption peak at 320 nm in the UV-Vis spectrum. The fluorescence spectra showed that the optimum excitation and emission wavelengths of the N-GQDs were 350 nm and 450 nm, respectively. The aqueous solution of N-GQDs appeared pale yellow under daylight and emitted blue fluorescence under a 360 nm UV lamp. The N-GQDs fluorescent probe showed a good linear response (R²=0.99) to ammonia-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, analysis of the stability, repeatability, and anti-interference properties of the fluorescent probe indicated that it possessed good stability and anti-interference capabilities against Na⁺, Hg⁺, Ag⁺, K⁺, Pb²⁺, Ca²⁺, Cd²⁺, Cu²⁺, and NO₂^-. However, repeated addition of hydrochloric acid caused irreversible damage to the surface groups of the N-GQDs, leading to poor repeatability. The response mechanism of the N-GQDs fluorescent probe to ammonia-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 exhibit electron-accepting properties. And the pyrrole nitrogens doped into the GQDs carry a slight positive charge. Ammonia acts as a typical electron donor due to the lone pair of electrons on the nitrogen atom. Therefore, it is inferred that photo-induced electron transfer occurs 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 ammonia-N in drinking water, tap water, 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%. Experimental results demonstrated that the N-GQDs fluorescent probe has potential applications in the detection of ammonia-N in water.