A quantitative determination method of the total flavonoids in Olea europaea L. leaves using rutin as a standard substance was developed. Two coloration systems including Al(NO3)3-NaNO2-NaOH and AlCl3 were both used. The latter was selected as the optimal determination method by comparing with the results. The main interference of the matrix was discussed. The result showed that the AlCl3 coloration method is simple, accurate and reproducible with good linear relationship, and it can be looked as a reference method for the determination of total flavonoids in herbal materials or natural products.