<p>Cysteine, one of the twenty proteinogenic amino acids, is distinguished by its unique sulfhydryl (–SH) functional group, which endows it with distinctive biochemical properties. In this work, nitrogen-doped carbon quantum dots (N-CQDs) were synthesized through a microwave-assisted strategy, with citric acid serving as the carbon source and urea as the nitrogen source. The as-prepared N-CQDs were employed for the detection of cysteine in liquid samples via an “off-on” fluorescence signaling mechanism. The fluorescence of N-CQDs was significantly quenched by Fe³⁺, whereas the subsequent addition of cysteine induced efficient fluorescence recovery. On the basis of this “off-on” fluorescence response, a highly selective, sensitive, and facile sensing strategy for the determination of cysteine was developed. Under optimized conditions, a good linear correlation was observed between cysteine concentration and relative fluorescence intensity over the range of 0–1.2 mmol·L⁻¹, with a detection limit of 10.49 µmol·L⁻¹. The calibration equation can be expressed as (F-F₀)/F₀ = 0.6496C_cys + 0.0475 (R² = 0.9932). This N-CQDs/Fe³⁺ fluorescence sensing system demonstrates considerable potential for practical cysteine determination in honey and aqueous samples.</p>