The invention relates to a preparation and technical method of a novel reagent (kit) required for detecting tiny RNA (miRNA) used in the field of biomedicine, and application thereof to the fields of clinical medicine, biology, molecular biology and the like. The invention belongs to the design, preparation and technical method application of the experiment reagent which uses specially designed primers and synthesized MGB probes for detecting and identifying mature miRNAs on the basis of a fluorescence quantitative PCR method. Compared with other methods, the technical method established by the invention has the characteristics of high sensitivity, high specificity, high throughput detection capability, short time consumption, relatively simple operation and the like. Thereby, the invention relates to the preparation of the novel miRNA detection reagent or reagent kit, the establishment of the technical method, the basic research of the reagent or the reagent kit, and the relevant prevention, treatment and research in clinical medicine such as malignant tumor, cardiovascular system diseases, nervous system diseases and the like.