The invention belongs to the technical field of molecular biology and particularly relates to a tobacco 25 S RNA (25S ribosomal RNA) reference gene sequence, a cloning method and application of the method. Part of the nucleotide sequence of the obtained 25 S RNA gene can be used as the reference gene used for researching gene expression analysis of tobacco functional genes or different structures in different growth and development stages and under different hormone treatment conditions. A real-time fluorescent quantitative PCR primer designed through the reference gene is high in amplification specificity, and stability, reliability and repeatability of tobacco gene expression analysis and research can be improved.