Abstract: At room temperature, 5 mL of ofloxacin solution (4.0×10-6mol·L-1) reacted with 5 mL of alizarin red solution (8.0×10-6mol·L-1) for 30 min to form a stable charge-transfer complex, which showed fluorescence. The maximum excitation wavelength of the complex was 303 nm, and the maximum emission wavelength was 508 nm. The composite ratio of ofloxacin and alizarin red in the complex was 1∶1. Based on that, a method of fluorescence spectrometry was established for the determination of ofloxacin. A linear relationship was found between the fluorescence intensity and the concentration of ofloxacin in range of 2.0×10-7-4.0×10-6mol·L-1, with detection limit of 9.1×10-9mol·L-1. Recovery rates obtained by standard addition method were in the range of 96.0%-101% and RSDs (n=5) was less than 2%. The method was applied to the determination of ofloxacin in pharmaceutical formulation, giving results in consistent with the marked values.