The LMX fluorescence properties at various pH of media were studied. The experiments indicated that LMX fluorescence emission wavelength is red shifted 40 nm at pH value of 3.3. The background interference was eliminated effectively. Under this condition, a new method for the determination of lomefloxacin in human urine by derivative-synchronous fluorescence has been developed. The linear equation of LMX can be obtained as dF/dλ = - 6.642c - 24.143, of which the correlation coefficient is 0.999 6. When concentrations of LMX were within the range of 0.35-28.10 mg*L-1 ,there exists a good linear relationship between the concentration and fluorescence intensity of LMX. The detection limit was 0.35 mg*L-1 .The average recoveries of LMX were 97%-104% and the RSD were 0.83%-1.62%.