The work aims to provide theoretical support for development of prevention and control measures for post-harvest disease of grape by isolation and identification of pathogenic bacteria of fresh grape black spot disease. The article used four table grapes (MANAI grape etc.) during storage as testing material to separate and purify pathogenic bacteria which caused disease of black spot disease. The colonial morphology on PDA medium and microscopic characteristics of spores and hyphae morphology of every quasi strain was analyzed. The epidemiological characteristics on berry inoculated with and without wound. ITS universal primers were applied to amplify the genomic DNAs of pathogenic bacteria and sequence the fragments. Three priori pathogenic pseudo strains (identified as A, B and C) were isolated and the frequency of them was detected as 75.0%, 32.0% and 42.11% in sequence. Grape, inoculated with and without wound with three pseudo strains, were pathogenic, and there were higher pathogenicity in those with wound than those without wound. By comparison with the fungus identification manual, the article preliminarily judged that pseudo strain A, B and C was Alternaria, Botrytis cinereal and Phomopsis, respectively. The sequence analysis of rDNA-ITS confirmed that the sequence similarity between strain A and KJ489375.1 and GU190188.1 was 99%. The main pathogenic fungus was confirmed to be Alternariaalternata. The main pathogen causing the black spot disease in fresh grape during storage and transportation is Alternaria alternata.