Background To construct and analyze the differentially expressed gene library in the SD rat's brain of Parkinson's disease.Methods The lateralization Parkinson's disease model of SD rat was established by stereotaxis injection of 6-OHDA in lateralization corpora striatum. Suppression subtractive hybridization was utilized to isolate the cDNA fragments in both control and experimental groups. Then the cDNA fragments were directly bound to pMD18-T to set up the subtractive library. Amplification of the library was carried out by transformation of E. coli with high voltage electroperforation. Ninety five positive bacteria clones were randomly picked up and identified by colony PCR.Results The amplified library contained more than 1500 positive bacteria clones. Ninety-five clones were analyzed randomly by colony PCR. And there were 81% clones contained 100 ～600 bp DNA fragments. They might be the cDNA fragments of differentially expressed genes of Parkinson's disease.Conclusions A differentially expressed genes subtracted cDNA library of Parkinson's disease of SD rat was constructed successfully with SSH and T/A cloning techniques. The library is efficient and it was useful in screening and cloning new and specific differential expression genes of Parkinson's disease.