Based on the self-ordered ring (SOR) fluorescence microscopic imaging technique on a hydrophobic glass slide with Zn2+ and cetyltrimethyl ammonium bromide (CTMAB) as sensitizer, and poly (vinyl alcohol) -124 (PVA-124) and NH3-NH4Cl (pH 10.00) as the medium, a method has been developed for determining sparfloxacin (SPFX) concentrations in chicken serum and residues in chicken tissues and manures. When the droplet volume was 0.20 μL, SPFX was determined in the range of 1.38×10-13～2.03×10-12 mol·ring-1 (or 6.90×10-7～1.02×10-5 mol·L-1), and the limit of detection (LOD) was 14 fmol·ring-1 (or 6.90×10-8 mol·L-1). The recoveries of SPFX at all different spiked levels are in the range of 90.74%～106.61% when the methanol or acetonitrile were used as extracting agent, respectively, and the relative standard deviations (RSDs) are less than 3.0%. This study expands the applied fields of SOR technique in drug concentrations and residues determination.