With agaric as carbon source and urea as nitrogen source, this paper has successfully synthesized nitrogen-doped carbon quantum dots (N-CQDs) by simple one-step hydrothermal method. A series of analysis have been used to make characterization of the physical, chemical structures and optical performance of N-CQDs. The fluorescence stability was studied by examining the ionic strength, pH value and storage time and other conditions. The cytotoxicity and bacterial toxicity of N-CQDs were applied to biological imaging. Considering the quenching effect of 2,4-dinitrophenol (2,4 DNP) on the fluorescence generated by N-CQDs, N-CQDs have been used as fluorescence probe in detection of 2,4-DNP where N-CQDs showed high sensitivity, selectivity and strong disturbance resistance to 2,4-DNP based on the quenching mechanism of transfer of resonance energy. It can also be concluded that agaric-based N-CQDs can be used in bio-imaging due to a good inhibitory effect on but low toxicity to bacteria. Under the optimal experimental conditions, the probe has presented a good coefficient of determination (R2 = 0.9969) and a low limit of detection (15.78 nM). This method has been successfully applied to determine 2,4-DNP in environmental samples and provided a new idea for developing green and natural composite carbon materials.