The fluorescence spectra and binding reaction of neutral red with nucleic acids have been studied. At pH=4.5, a new method for the determination of trace nucleic acids based on fluorescence quenching measurement was established. When 8.0×10 -6 mol/L NR is employed, the linear ranges of the calibration graphs are 0.08-2.0 μg/mL for calf thymus DNA(ctDNA), 0.12-2.2 μg/mL for fish sperm DNA(fsDNA) and 0.30-1.4 μg/mL for yeast RNA(yRNA), respectively. The corresponding detection limits are 30, 33 and 66 ng/mL, respectively. The real samples were analyzed satisfactorily.