Abstract: Malachite green is not only a dye, but also a fungicidal, bactericidal and antiparasitic drug, but excessive consumption for a long time could cause carcinogenic effect, so it was banned from using in the field of pollution-free aquaculture. However, malachite green has therapeutic effect on the water mould of fish body and eggs, which could also effectively prolong the life of scale damaged fish in the transportation process, so the use of malachite green in aquatic products is difficult to prohibit in China. Many efforts have been devoted to develop other detection methods, such as surface enhanced Raman spectroscopy (SERS), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry, immunoassay, electrochemical method, colorimetry, molecular imprinting and fluorescence method, but there are scarce reports on the application of fluorescence quenching sensor for quantitative analysis of malachite. In this paper, nitrogen sulfur-doped graphene quantum dots was successfully prepared, and characterized by transmission electron microscopy, XPS and fluorescence spectrometry. The results showed that there were C、O、N、S elements in the nitrogen sulfur-doped graphene quantum dots, of which particle size was 2-8 nm, the maximum fluorescence emission wavelength was 449 nm, and the exciting wavelength was 370 nm. When different concentrations of malachite green were added into the nitrogen sulfur-doped graphene quantum dots solution, obviously decreasing of the fluorescence intensity of nitrogen sulfur-doped graphene quantum dots could be observed, and there was a good linear relationship between the concentration of malachite green and variety of the fluorescence intensity of nitrogen sulfur-doped graphene quantum dots (F₀/F). A fluorescence quenching sensor for quantitative analysis of malachite green was established, the variety of fluorescence intensity of nitrogen sulfur-doped graphene quantum dots was proportional to the concentration of malachite green in the range of 8.22-13.7 nmol/L, the limit of detection of 4.830 nmol/L(1.762 ng/mL). Quenching mechanism of this quenching system has been also discussed, there was obvious difference between before and after addition, indicating that malachite green has been compounded with nitrogen sulfur-doped graphene quantum dots. Variable temperature experiments indicated that it is a static quenching process with the formation of compound between nitrogen, sulfur-doped graphene quantum dots and malachite green, the stability of this compound decreased with the increasing of temperature, and the slop of regression linear decreased. Particularly, quantitative analysis of water samples indicated that this method could be more widely applied to similar samples. This was a simple, fast and low cost method, which could be used for quantitative analysis of malachite green.