According to the principle that NO^－₂ is able to enhance the fluorescence intensity of 4-hydroxycoumarin, a spectrofluorometric method for determination of nitric oxide in biological sample was developed. The nitrite concentration in the range of 2×10^-5 mol/L to 2×10^-8 mol/L in the sample can be measured. The method has been applied to determine the level of NO in cortex and hippcampus, cultural supernatant of macrophage actived by LPS and serum in rat.