Terbium(III) forms highly fluorescent complex with norfloxacin. The presence of surfactant can enhance greatly fluorescence intensity. In this paper, the fluorescence characteristics of complex in presence of surfactant has been investigated. It was found that norfloxacin could form stable complex with terbium in the pH 6.5–8.5 media. The fluorescence intensity of complex in SLS media is three folds more than complex itself. A new micelle enhancement spectrofluorimetry for the determination of norfloxacin has been developed. The detection limit of proposed method is 0.017 μg/ml. It has been satisfactory for the determination of trace norfloxacin in serum with 95.2%∼101.2% recoveries.