Abstract: In order to establish a new process for the preparation of Rhodococcus ruber extract, by taking Rhodococcus ruber FIM-12 as the experimental material, the preparation conditions of Rhodococcus ruber extract were optimized by using the single factor test, and the main components such as lipoic acid, total polysaccharide and amino acids were further analyzed. The results showed that: the preparation yield of Rhodococcus ruber extract was as follows: 3.97 g Rhodococcus ruber extract could be prepared from 100 g wet cells, which contained 17.48% lipoic acids and 44.1% polysaccharides. It contained 1 kind of glucosamine (cell wall characteristic amino acid) and 18 kinds of amino acids (including 8 kinds of essential amino acids), with relatively rich species. The research results could provide reference for the further development and utilization of Rhodococcus ruber extract.