The complex of pefloxacin-La3+ was formed in the pH 5.6 HAc-NaAc buffer. Its fluorimetric intensity was 1.5 times that of the pefloxacin, and the excitation and emission peaks of the complex were at 276 and 440 nm, respectively. The new fluorescence method was developed for determining pefloxacin in serum based on the characteristics. The content of pefloxacin in serum was 4.79 mg x L(-1), the RSD and recovery were 6.3% and 95% respectively. The determination range was 0.04-2.0 mg x L(-1) with the detection limit of 2.8 microg x L(-1). The proposed method and UV-Vis photometric method were both applied to determining the pefloxacin in the same capsules with satisfactory results.