Rice sheath blight is one of the common soil-borne diseases of rice. To develop a biological rice breeding substrate to prevent and control rice sheath blight and promote rice growth, 5 rhizosphere growth-promoting bacteria with nitrogen fixation and phosphorus removal functions were selected, including LY5 (Bacillus subtilis), LY11 (Bacillus amyloliquefaciens), X2 (Pseudomonas moraviensis), X3 (Bacillus safensis) and X8 (Pseudomonas chlororaphis). Based on the determination of the inhibition rate, the 5 bacteria were mixed with pathogenic bacteria of rice sheath blight, and then the functional biological rice breeding substrates were developed on the basis of the most suitable substrates screened in the laboratory, and a pot experiment was conducted to verify the effects of the developed 5 substrates on the incidence rate of rice sheath blight, growth of rice seedlings and in vivo resistance-related enzymes in rice by comparing with a commercial rice breeding substrate (CK). The results showed that the 5 rhizosphere growth-promoting bacteria all had the antagonistic effect on the pathogen of rice sheath blight with the inhibition rate from 35.31% to 40.20%. Compared with CK, the 5 rhizosphere growth-promoting bacteria all reduced the incidence rate of rice sheath blight, among of which, LY11 treatment significantly reduced the incidence rate by 89.32% and increased the number of emergence per plate by 110.41%. The 5 rhizosphere growth-promoting bacteria increased rice stem diameter, underground dry matter weight of 100 plants, dry matter weight of 100 plants, root activity, total root surface area, average root diameter, total root volume and root tip number, among of them, compared with CK, LY11 treatment significantly increased the above indexes by 3.13%, 4.21%, 4.97%, 68.02%, 26.77%, 16.67%, 44.44% and 7.82%, respectively. Compared with CK, the 5 rhizosphere growth-promoting bacteria significantly promoted the uptake of nitrogen, phosphorus and potassium by rice seedlings, increased significantly the activities of superoxide dismutase, peroxidase and catalase and the content of malondialdehyde in rice seedlings by 27.08%-122.72%, 16.09%-48.15%, 53.33%-233.85% and 110.24%-511.71%, respectively. Therefore, the 5 rhizosphere growth-promoting bacteria have good antagonistic effect against rice sheath blight pathogens and Bacillus amyloliquefaciens has the best comprehensive effect, it lays a theoretical and practical basis for developing the high efficient biological rice breeding substrate with growth-promoting and bio-control functions.