Abstract: In order to identify the pathogenic bacteria causing walnut rot disease, the pathogenic bacteria were identified through tissue isolation and pathogenicity tie-back, and the species identification was completed based on morphological characteristics combined with molecular biology. Furthermore, the effects of three types of immune-inducers spray as well as five types of biologicals and two types of adjuvants scraping were compared on walnut rot. Fifteen strains were isolated from 14 diseased branch samples, of which 14 strains were identified morphologically and molecularly as Cytospora chrysosperma, and one strain was Cryptosphaeria pullmanensis. Therefore, the main causal agent of walnut rot disease in Hetian County was C. chrysosperma. Among the three kinds of immune-inducing antagonists, 0.5% chitosan AS 200× and amino-oligosaccharide AS 500× showed better-inducing effects of 51.43% and 45.47%, respectively. Among the biological agents, the control effects of 100 billion CFU/g Bacillus amyloliquefaciens WP 700× + 99% SK Enspray 99 EC 200× and 3% Zhongshengmycin AS 500× + 99% SK Enspray 99 EC 200× were better, 92.31% and 84.61%, respectively. This study clarified the pathogen of walnut rot disease in Hetian County and provided a reference for the biological control of walnut rot disease in the field.