A new method of determinating doxycycline residues in milks and content in serum,table and injection samples was established with Self-ordered ring fluorescence microscopic imaging technique. In the presence of hexahydropyridine and poly (vinyl alcohol)-124, Zn2+-doxycycline (DC) system can form a SOR on the hydrophobic glass slides surfaces based on the capillary effect. The maximum fluorescent intensity (Imax) at central ring belt was found to be proportional to DC content. when the droplet volume is 0.5μL, the present SOR method can be used to determine DC in a range of 4.54×10-15–6.81×10-13 mol•ring-1, and the limit of detection (LOD) with a threefold signal to noise ratio (S/N = 3) was 4.54×10-15 mol•ring-1 (2.17×10-8 mol•L-1). With the present method, the residues in milks and content in serum, tablet and injection samples were satisfactorily detected with recoveries of 96.3-102.0% and RSD of 1.4-2.2%, respectively, indicating that the method is reliable and practical.