The fluorescent carbon dots were successfully synthesized by simply heating the mixture of lactose and Na OH solution. The as-synthesized carbon dots had been systematically characterized by fluorescence, Fourier transform infrared(FTIR), high resolution transmission electron microscopy(HR-TEM) and ~(13)C NMR. Since the fluorescence of the carbon dots was efficiently quenched by folic acid, the carbon dots were employed as selective fluorescence probes for detecting folic acid, depending on the formation of hydrogen bond among the functional group of folic acid(–OH, –COOH and –NH_2) and –OH and –COOH of the carbon dots. Moreover, the decrease of fluorescence intensity was capable of detecting folic acid in a linear range of 6×10~(-5)–8×10~(-8) mol/L with a detection limit of 1.2×10~(-9)mol/L at a signal-to-noise ratio of 3, suggesting a promising assay for folic acid. Significantly, the practicability of this fluorescence probe to assay folic acid in human urine samples was further evaluated. The fluorescent carbon dots were successfully synthesized by simply heating the mixture of lactose and NaOH solution. The as-synthesized carbon dots had been systematically characterized by fluorescence, Fourier transform infrared (FTIR), high resolution transmission electron microscopy (HR-TEM) and ~ (13) C NMR. Since the fluorescence of the carbon dots was efficiently quenched by folic acid, the carbon dots were employed as selective fluorescence probes for detecting folic acid, depending on the formation of hydrogen bond among the functional group of folic acid ( -OH, -COOH and -NH_2) and -OH and -COOH of the carbon dots. Moreover, the decrease of fluorescence intensity was capable of detecting folic acid in a linear range of 6 × 10 -5 - 8 × 10 ~ (-8) mol / L with a detection limit of 1.2 × 10 -9 mol / L at a signal-to-noise ratio of 3, suggesting a promising assay for folic acid. Significantly, the practicability of this fluorescence probe to assay folic acid in human urine samples was further evaluated.