[Background] Pestalotiopsis trachicarpicola is a new pathogen causing black spot disease of Eucommia ulmoides. There is no report that biocontrol bacteria had antagonistic activity on it. [Objective] The best Bacillus sp. antagonistic activity against P. trachicarpicola DZHBB-1 was isolated and selected from healthy leaves of E. ulmoides, the rapid and accurate identification of this strain was achieved by using the 16S rRNA gene binding protein-encoding gene. [Methods] The strains were isolated from healthy E. ulmoides leaves by dilution separation method, screened by sputum experiments and growth rate methods, combined with 16S rRNA, gyrA and gyrB gene sequences to construct phylogenetic tree analysis. The target strains were identified by its morphological characteristics, physiological and biochemical properties. The pot experiment was carried out to verify its control effect on black spot of E. ulmoides. [Results] A total of 62 strains of Bacillus were isolated and 14 strains with good biocontrol effect on pathogens were screened out. The results of rescreening showed that the strain J1 had the best antagonistic activity and was stable, the inhibition rate reached 66.67%. Phylogenetic tree analysis of 16S rRNA, gyrA and gyrB gene sequences showed that strain J1 was B. amyloliquefaciens. In the pot experiment, the strain could effectively prevent the occurrence of black spot disease of E. ulmoides and the control effect exceeded 57%. [Conclusion] This strain has the potential to prevent and cure the related plant diseases caused by P. trachicarpicola. 16S rRNA, gyrA and gyrB genes are combined to identify Bacillus sp., which can provide a reference method for the rapid and accurate identification of Bacillus sp. and its related species.