A cost-effective and environmentally friendly method was formulated for rapid dopamine (DA) detection that was based on the fluorescence (FL) quenching of carbon quantum dots (C-dots). Upon adding DA to the C-dots’ solution, we noticed a regular reduction in their fluorescence intensity. The effects of pH, amount of C-dots, reaction temperature and time on the determination of DA were investigated. Under the optimized experimental conditions, trace amounts of DA could be analyzed. Furthermore, dopamine hydrochloride injection and human urine samples with and without spiked DA were analyzed using the developed sensing system. The procedure was validated following the guidelines of the European Medicines Agency (EMA) in terms of the following: calibration range (0.3–100 μM), linearity (R2 = 0.9991), limit of detection (LOD) (93 nM). Recoveries of dopamine with spiked samples at three different levels were between 95.0 and 105.9%, and the relative standard deviations (RSDs) were within 2.68% (n = 6). This method is simple and suitable for the determination of dopamine in pharmaceuticals and human urine for clinical application. Compared with previous reports, the proposed method offers great advantages including ease of C-dot sensor preparation (one-pot synthesis), environmentally friendly sample preparation by using either water or phosphate buffer solution only, a short response time and selectivity.