Saffron is one of the world's most precious medicinal herbs and often found to be adulterated with other cheaper materials. The chemical compounds in Crocus sativus L. such as crocin, picrocrocin also exist in other plants, which makes the chemotype-driven analysis not ideal for the quality control of saffron. Herein, we developed a rapid authentication method for saffron in crude drugs by the site-specific PCR. In order to realize fast high-throughput analysis, a one-tube identification approach was further established by using a universal fluorescent dye to detect the PCR products. In addition, this method was also applied to the authentication of saffron in a processed herbal medicine "Er shi wu wei shan hu wan" which consists of twenty-five kinds of medicinal materials including plants, minerals and animals. Additionally, this method was also proved to be with high specificity and repeatability. The flexibility of choosing different primers also made this method versatile for other medicinal materials.