Abstract: The critical gene phlD for 2, 4-DAPG biosynthesis from Pseudomonas fluorescens was cloned and expressed in E. coli BL21(DE3)/pET28a(+)/phlD recombinant strain. The gene of phlD was amplified using Pseudomonas fluorescens 2P24 genome as template. The PhlD of 42000Da fusion protein was induced and expressed in E.coli BL21(DE3)/pET28a(+) system. The phlD gene sequence was analyzed, the senior structure of the protein coded by the phlD also forecasted. PhlD was one of polyketide synthase of type-Ⅲ, and the function of PhlD was similar to the polyketal synthase and acyltransferase. The fermentation liquid of recombinant strain was detected, and synthesis of the phloroglucinol was implemented.