Nitrogen-doped blue fluorescent carbon dots (N-CDs) were synthesized by one-step hydrothermal method using citric acid as the carbon source and urea as the nitrogen source. The morphology and structure of N-CDs were characterized by high resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), ultraviolet spectroscopy and infrared spectroscopy. The results showed that the N-CDs were well-dispersed spherical particles with an average particle size of 2.90±0.30 nm. Fe3+ could combine with the nitrogen-containing groups on the surface of N-CDs, leading to their fluorescence burst, while pesticide amitrole (AMT) has stronger affinity with Fe3+. When AMT is present in the N-CDs/Fe3+ coexistence system, Fe3+ could be desorbed through AMT competition and the fluorescence of N-CDs can be restored. Based on the above principle, an “on-off-on” fluorescence detection method was developed to detect amitrole in nitrogen-doped carbon dots. The linear range of the method was 0.40~40.00 μg/mL, the correlation coefficient was 0.999 6 and the detection limit was 0.18 μg/mL.Other ions and pesticides showed little interference on the method, thus demonstrating good specificity. The method was also applied for AMT detection in real samples with the recoveries of 100.01%~115.43%, which is satisfactory and has good application prospect.