This application relates to a sweetening composition and a preparation method and use thereof. The method includes steps of obtaining mesophyll fragments of Rubus suavissimus S. Lee, extracting with water as a solvent, removing phenolic hydroxyl-containing components, concentrating, purifying, and water-phase crystallization to obtain a sweetening composition. The sweetening composition is white in color, with unobvious bitterness astringent taste. The sweetening composition contains 50% to 99% of Rubusoside based on a dry weight, and has an absorbance of less than 0.4 at a wavelength of 270 to 370 nm after being dissolved and prepared into an aqueous solution (with a solid content of 1%, w/w). By removing bitter glycosides and phenolic hydroxyl-containing components, this application makes the flavor of the sweetening composition better. In the preparation process of the sweetening composition of the this application, only purified water is used and no organic solvents are used.