The present study obtained a PKS Ⅲ gene from the transcriptome of the lichenizedfungi Nephrmopsis pallescens. We first cloned fulllength cDNA of a PKS Ⅲ gene through the method of RTPCR, and named it NpPKS3, then we had a trial of NpPKS3 gene expression in the different media, to acquire the optimal media culturing the lichenforming fungi N. pallescens, apply some basis to further explore the function of NpPKS3 gene, and simultaneously offer some necessary material to the heterologous expression of polyketide compounds in the lichenizedfungi N. pallescens. The results showed: (1) the full length of NpPKS3 gene(GenBank ID: MF351559) has 1 335 bp, codes 444 amino acid, its transcriptional product could be polyketide synthase of N. pallescens (NpPKS3), and indwells cytoplasmic matrix. (2) The phylogenetic analysis revealed that the NpPKS3 amino acids sequence and the csyB of polyketide synthase Ⅲ clustered into one clade, therefore we could speculate it might be csyB. (3) We detected that the conditions of the NpPKS3 gene expression in the different media by OSMAC(one strain, many compounds) strategy. The result showed it is the best expression ability of NpPKS3 gene in BMG medium, and the weakest in BD medium.