[Objective] In order to isolate and screen a high-affinity siderophore-producing bacteria from calcareous soil, which has a significant effect on the pathogen. [Methods] High-affinity siderophore-producing strains were screened by chrome azural S (CAS) assay. Then, we take the methods of morphological observation, biochemical and physiological characteristics, 16S rRNA gene sequence and phylogenetic analysis to identify the bacterial species. Based on the results obtained from strain’s screening and identification, antagonism between strains and pathogenic bacteria was researched by dual culture on potato dextrose agar. [Results] Siderophore-producing bacteria HMGY6B identified as Pseudomonas was isolated and screened out from the ryegrass (Lolium perenne L.) rhizosphere soil, which produced Catechol-type siderophore had the significant effects on cucumber gray mold (Botrytis cinerea). The inhibition ratio was up to 91.2% under low iron conditions (0.16, 2, 5, 10 μmol/L FeCl3). But unfortunately, it decreased to 30.2% under 50 μmol/L FeCl3 and only 5.5% under Fe-replete conditions (100 μmol/L FeCl3). [Conclusion] The srtain HMGY6B can be used to the exploitation and development of compound disease-resistant manure.