In the Tris-HCl buffer solution, europium ion combined with pipemidic acid (PPA) to form a Eu-PPA complex. The fluorescence intensity was dramatically enhanced when herring sperm DNA was added in the Eu-PPA system. Furthermore, the fluorescence intensity was increased linearly with the concentrations of added DNA within a proper range. Based on this, a simple new method for the determination of herring sperm DNA was developed. Time-resolved fluorescence excitation and emission spectra and the fluorescence lifetime of the system were studied in detail and compared with that from conventional fluorescence method. The experimental results indicated that the time-resolved fluorescence method was superior over the conventional fluorescence method. The reaction conditions, measurement parameters and the adding order of reactants were optimized. The best concentration of both europium ion standard solution and PPA standard solution was 1.00×10-5 mol·L-1 in the Tris-HCl buffer solution of 0.005 mol·L-1 with the optimal pH value of 7.2. A detection limit (3 s) of 0.03 mg·L-1 was obtained with a RSD of 0.3％ (4.0 mg·L-1, n=11). The linear relationship between ΔI=89.58ｃ(mg·L-1)+0.920 5 and the regression factor of 0.999 6 was obtained in the range of 0.1-6.0 mg·L-1. This method was applied to the determination of DNA in synthetic samples. The results and recoveries of spiked standard solution in the sample were satisfied.