Mg2+-sensitized metacycline fluorescence microscopic imaging technique was applied to detect the raw fresh milk of four cows breeding farms in the Miyun County of Beijing based on the capillary effect of solvent on solid supports. In the presence of NH3-NH4Cl buffer solution (pH 9.99) and PVA-124, Mg2+ and metacycline can form a strong fluorescence complex of 1∶1, and Mg2+-metacycline complex can form an SOR on the hydrophobic supports with the diameter of 0.93 mm and its ring belt width of 26.2 μm. By measuring the fluorescence intensity of the ring, the quantitative analysis of metacycline was achieved with the detection limit (3σ) of 8.8×10-14 mol·ring-1 (1.8×10-7 mol·L-1) and linear range of 2.2×10-13-3.6×10-12 mol·ring-1 (4.4×10-7-7.2×10-6 mol·L-1) when 0.50 μL droplet was spotted. This method has been satisfactorily applied to the determination of metacycline in the raw fresh milk samples with the recovery of 93.8%-108% and RSD less than 4.3%