The method of CTMAB-Al3+-OFLX ternary complex fluorescence microscopic imaging technique was established for the determination of ofloxacin based on the capillary effect of solvent on solid supports, and the concentration in the serum after the chicken was burdened with ofloxacin tablet, the concentration in the human urines and the percentage composition in the honeies, ofloxacin tablets and eye-drops were measured with satisfaction, respectively. In the presence of pH 9.50 NH3-NH4Cl buffer solution and PVA-124, CTMAB-Al3+-OFLX ternary complex can form a self-ordered ring on the hydrophobic supports with the diameter of 1.63 mm and its ring belt width of 50 μm. When a 0.20 μL droplet was spotted, the fluorescence intensity of the ring had a favorable linear relation (r=0.999 2) with the drug concentration in the range of 3.30×10-13~1.65×10-12 mol·ring-1 (0.60~2.98 mg·L-1) and the limit of detection can reach 4.10×10-15 mol·ring-1 (7.41 μg·L-1) with three times of signal to noise ratio. This method has been applied to the average concentration of ofloxacin in the chicken serum with the recovery of 96.4%~101.2% after two hours of being burdened with ofloxacin tablet. Then the technique was applied to the determination of ofloxacin in the three healthy volunteer’s urines after oral administration with recovery of 98.2%~106.1%. It was found that the concentrations of ofloxacin in urines were the highest after three hours of taking medicine; the result was similar to reports in the literature. The residues of ofloxacin in three different honey samples were satisfactorily determined with the recoveries of 98.2%~106.1%, and RSD was less than 2.3%. The contents of active constituent in tablet samples and eye-drops sample were determined with recoveries of 93.5%~101.5% and 95.8%~104.2%, and RSD was 3.5% and 3.6%, respectively, which were similar to marked values.