The invention discloses an internal reference gene for detecting transcriptive expression of Muscari Botryoides Mill gene. The nucleotide sequence of the internal reference gene is represented by SEQ ID NO.1, and the internal reference gene can meet requirements of qRT-PCR detection of the transcriptive expression level of the Muscari Botryoides Mill gene and improves the accuracy of the detection of the transcriptive expression level of the Muscari Botryoides Mill gene by using a qRT-PCR method. The invention also discloses an application of the internal reference gene in the qRT-PCR detection of total RNA of Muscari Botryoides Mill. A primer pair ACT T1-5077-s-2 provided by the invention can be used to obtain the high and stable expression internal reference gene, and avoids the problem of low detection accuracy of the mRNA expression of the Muscari Botryoides Mill gene, caused by poor mRNA expression stability of internal reference genes.