A novel ratiometric fluorescence probe for the determination of levofloxacin hydrochloride (LVF) was established based on fluorescence resonance energy transfer (FRET) between 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and acridine orange (AO). Under 310 nm excitation, the fluorescence spectra of TMB at 350～500 nm overlap with the absorption spectra of AO in the pH 5.0 NaAc-HCl buffer solution. Accordingly, the FRET system is constructed with TMB as the energy donor and AO as the energy receptor. According to the energy transfer theory, the FRET efficiency of the system is 62.5%, and the distance between the donor and receptors is 2.17 nm, further indicating that FRET occurs between TMB and AO. When LVF is added, TMB transfers the fluorescence energy to LVF, which transfers the energy to AO as a donor. LVF acts as a bridge between TMB and AO. LVF transfers the absorbed fluorescence energy of TMB to AO, resulting in a significant decrease in the fluorescence intensity of TMB and a significant increase in the fluorescence intensity of AO, thus improving the FRET efficiency of the system. Under the optimal experimental conditions, there is a good linear relationship between the ratio of F546 nm to F402 nm and LVF concentration in the 2～80 μmol·L-1 range. The linear regression equation is F546 nm/F402 nm=87.916c+3.108, the linear correlation coefficient is 0. 9993, and the detection limit (LOD) is 15.7 nmol·L-1. Some common cations (K+， Mg2+， Ca2+， Cu2+， Mn2+， Zn2+， Co2+， Ni2+， Cr3+, etc), anions (F-， Br-， NO-3， IO-3， CO2-3， SO2-4, etc), sugars (glucose, sucrose and starch), drugs (glutathione, ascorbic acid and isoniazid) and several amino acids (glycine, leucine, cysteine, etc) do not interfere with the determination of levofloxacin, indicating that the ratiometric fluorescent probe has high selectivity for LVF. The method was applied to determine LVF in commercial pharmaceutical preparations with a recovery of 93%～97%. The ratiometric fluorescence probe has great potential in clinical application for the detection of LVF, which provides a good theoretical basis for the development of a simple, selective and sensitive sensor for the determination of LVF in pharmaceutical preparations, and a certain method guidance for improving the safety and rationality of LVF inclinical medication.