The invention discloses a preparation method of a new bacterial strain t16 of nitrogen-fixing bacterium (Rhizobium sp.) for astragalus membranaceus, which is collected in China General Microbiological Culture Collection Centre on September 18, 2014, with a collection number of CGMCC No. 9576. The invention describes the morphological characteristics and the physiological and biochemical indexes of the new bacterial strain t16 of nitrogen-fixing bacterium (Rhizobium sp.) for astragalus membranaceus, a 16S rDNA sequence determination result and the like in details. A nitrogen-fixing test indicates that the fermentation liquor of the new bacterial strain t16 of nitrogen-fixing bacterium (Rhizobium sp.) for astragalus membranaceus has a good nitrogen-fixing effect on astragalus membranaceus, and can effectively provide a natural nitrogen source need for the growth of astragalus membranaceus, thus providing a corresponding nutrition source for the natural quality of astragalus membranaceus.