Abstract: Penicillium griseofulvum D-756 was a mutant strain with high yield of griseofulvin. Revealing the expression of key genes in the biosynthesis of griseofulvin at the transcriptional level would be helpful to understand its gene function and to carry out the directional genetic modification of the strain. The primers were designed by using Primer3 and Primer BLAST. After the qPCR verification, the amplified products of the primers were single and the amplification efficiency was in the range of 90%-110%. Then, the expression stability of the four candidate reference genes TUBB, HISH3, RPS24 and 28S rRNA was evaluated by using the geNorm software. The candidate genes TUBB and RPS24, which were relatively stable in the evaluation results, were selected as the reference genes to construct a qPCR system suitable for Penicillium griseofulvum D-756. Last, this system was used to detect the relative expression of gsfA, the key gene for the biosynthesis of griseofulvin in Penicillium griseofulvum D-756, at different culture times, thus to provide a powerful tool for the further study of each gene function in the biosynthetic gene cluster of griseofulvin.