A nucleotide sequence having SEQ ID NO:1, a universal reverse primer having SEQ ID NO:2, a universal RT primer, a method for designing primer, and a miRNA detection method are provided. In the miRNA detection method, the universal RT primer is used for the cDNA synthesis of a miRNA sample and the universal reverse primer is used for cDNA molecule amplification in qPCR quantitative detection. The universal reverse primer sequence, the nucleotide sequence, and the design rules are used in the method for designing primer, so as to design a primer for qPCR quantitative detection.