Fluorescent quantitative PCR is one of the first choices for the methods in analyzing gene expression level. It is simple for operation, but how to ensure the credibility of the quantitative PCR results is still a problem to be tackled especially for the quantitative analysis of miRNA with only about 20 nucleotides. In this paper, the expression difference of miR408 gene in different tissues in rice was taken for instance. The new criteria and requirements of fluorescent quantitative PCR (RT-qPCR) were systematically optimized and summarized. The results from this study indicates that the concentration of primers plays a crucial role in optimizing the fluorescence qPCR detection system.