Abstract: Total RNA of 1~15 d red line callus of S.medusa was extracted by TRIZOL Reagent. cDNA library was constructed with SMART cDNA Library Construction Kit. The primary library has a high titer from 1.5×106 to 4×106, in which 98% clones are recombinant and the insert cDNAs are from 0.5 kb to 3 kb. The amplified library has a titer of 1011. The positive signals of CHS, DFR and SmP gene were detected by PCR in the amplified library, though SmP gene was expressed at low abundance . This high quality cDNA library provides a useful tool for the study of the molecular mechanisms of the secondary metabolism of the flavonoids of S.medusa.