Abstract: This work aimed at developing a fluorescence assay for rapid and sensitive determination of alkaline phosphatase (ALP) activity in fresh milk using nitrogen doped carbon dots (N-CDs) as fluorescence probe. Green-emissive N-CDs were synthesized by using ethylenediamine as the nitrogen source and catechol as the carbon source through a hydrothermal method. The obtained N-CDs were characterized by transmission electron microscopy (TEM), UV-vis spectroscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FT-IR) spectra. Control experiments were used to verify the feasibility for constructing the fluorescent assay using the N-CDs. Single-factor experiments were conducted to optimize the pH of working buffer, the concentration of disodium p-nitrophenyl phosphate (PNPP) and enzymatic reaction time for ALP detection. The results indicated that the as-synthesized N-CDs were successfully prepared with all the characterization results being consistent with those in previous works. The optimal working conditions for this assay were as follows: Tris-HCl (20 mmol/L, pH10) as working buffer, 1 mmol/L of PNPP as enzymatic substrate, incubation for 50 min. Under the optimal conditions, it was found that fluorescence intensity of N-CDs linearly corelated with ALP concentration from 0.01 to 25 U/L with correlation coefficients of 0.995. The linear regression equation was Y=15397.05X+70344.46 with a limit of detection (LOD) of 0.001 U/L (S/N=3). Recoveries for two kinds of fresh milk were in the range from 100.1% to 107.2%, and the coefficients of variations were less than 14.3%, which indicated that the proposed method had good accuracy and reliability. This study provides an accurate and efficient method for the detection of ALP in fresh milk.