A method utilizing nitrogen-doped and sulfur-doped carbon quantum dots (N, S-CQDs) as fluorescent probes for the rapid detection of Fe3+, L-ascorbic acid (AA), and alkaline phosphatase (ALP) was presented. The fluorescence intensity of N, S-CQDs nanoprobes can be rapidly and efficiently quenched by Fe3+ and based on the fluorescence "turn off-on" characteristic of N, S-CQDs nanoprobes, the fluorescence signals of the N, S-CQDs/Fe(3+)can be recovered after the addition of AA. By coupling a fluorescent nanoprobe to an enzyme and L-ascorbic acid-2-phosphate (AA2P), a green, simple, rapid and effective fluorescent analytical method for the determination of ALP was developed. The prepared N, S-CQDs showed high sensitivity and selectivity to Fe3+, AA and ALP with the detection limit of 0.42 mu M, 12.7 nM and 0.017 U center dot L-1 and their optimal concentration ranges were 10-600 mu M, 10-200 mu M, 0.18-54 U center dot L-1, respectively. The fluorescence quantum yield of N, S-CQDs (0.2 mg center dot mL(-1)) at 393 nm excitation wavelength was 4.41%. Additionally, the fluorescent nanoprobes have been employed to successfully measure ALP in serum samples. It is expected that the established method may offer a new approach for biomolecular detection in clinical diagnosis and pharmaceutical analysis.