In this paper, the effect of the cross-linking agent 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) on the optical properties of graphene quantum dots (GQDs) and the reasons are investigated in detail. GQDs were prepared by a hydrothermal method and reacted with EDC to obtain GQDs/EDC composites. The spectral properties of GQDs and GQDs/EDC were investigated. The effect of pH on the fluorescence of GQDs/EDC and its mechanism were investigated using PBS solution and artificial gastric juice samples. The experimental results show that EDC passives the surface defects of GQDs, making the fluorescence of GQDs increase rapidly in <1 min time and remain stable up to 20 min. Under different EDC contents the fluorescence intensity of GQDs/EDC is significantly enhanced by about 264 times compared to GQDs alone. The pH response experiments shows that GQDs/EDC had a linear response pattern of fluorescence and absorption intensity in the pH range of 1.75-4.01 and 4.01-9.28. Biocompatibility showed that the cell viability of human breast cancer cells was greater than 80% at sample concentrations of 25-300 µg/mL and remained at 74% even at high concentrations of 500 µg/mL; Finally, the detection of artificial gastric pH has high accuracy with relative standard deviation RSD ≤1.10%. The EDC-mediated fluorescence enhancement makes GQDs more advantageous in the fields of detection, sensing and imaging. Besides, the sensitive pH response characteristics of GQDs/EDC provide a good prospect for pH detection applications.