Abstract: A fluorescence spectroscopy method for the determination of ampicillin was proposed based on then weakened fluorescence quenching of nuclear fast red by Cu(Ⅱ) and the enhanced fluorescence intensity, which were caused by the complex formed between ampicillin and Cu(Ⅱ). The effects of buffer solution, the addition order of reagent, the concentration of nuclear fast red and Cu(Ⅱ), reaction temperature and time, and ionic strength were investigated. Under the optimized conditions, linear relationship between the enhanced fluorescence intensity and the mass concentration of ampicillin were in the range of 0.50-26 mg·L-1 with the detection limit (3s/k) of 0.40 mg·L-1. The method was applied to analyze ampicillin capsules, giving values of recovery in the range of 98.1%-101% and RSD′s (n=6) in the range of 1.7%-2.1%.