In this work, nitrogen-doped graphene quantum dots (N-GQDs) were synthesized by an efficient one-step hydrothermal using hydrated citric acid and urea as the carbon and nitrogen sources, respectively. The fluorescent quantum yield of the N-GQDs was up to 88.20 %, and the fluorescence lifetime was 12.29 ns. Based on the excellent fluorescence property, water-soluble and low cytotoxicity of N-GQDs, a novel fluorescent probe was fabricated and used for simultaneous detection of l-glutamic acid (l-Glu) and l-aspartic acid (l-Asp). Herein, Al3+ was used as a chelant agent for l-Glu shielding. The sensitive response of the probe towards l-Glu and l-Asp presented a wide concentration range with a low detection limit (S/N = 3). The fluorescent probe showed good selectivity, high stability and acceptable reproducibility in the application of l-Glu/l-Asp simultaneous detection in chicken soup. Meanwhile, the detection results of the fluorescent probe were correlated well with those obtained by amino acid analyzer.