Flavonoids, a large class of phenolic compounds widely exist in plants and vegetables, have been reported to be strong antioxidants and radical scavengers. Vitamin P is a kind of flavonoid glycoside known as rutin and has capacity in antiplatelet, antiviral, antihypertensive, and strengthing the capillaries of blood vessels, which are the results of its high radical scavenging activity and antioxidant capacty. A novel method for the determination of Vitamin P based on the enhancement of the intensity of lucigenin used as a chemiluminescent probe in basic medium is described. The present method allows the determination of Vitamin P over the range 4.58 - 305.27 μg/ml. The correlation coefficient of the working curve was 0.998. The relative standard deviation was 0.75% for 10 determinations of 7.5 X 10^-5 Vitamin P. The detection limit was 0.350 μg/ml. The effects of experimental parameters such as concentrations of lucigenin, KOH, H2O2 were studied. The method can be suitably applied for the determination of Vitamin P in pharmaceutical formulations.