In this research, the recombinant expression vector pBBR1-genta-RhlAB containing the rhamnosyltransferase gene (RhlAB)was modified via Red/ET homologous recombineering. Then, three recombinant expression vectors pBBR1-genta-Papra-RhlAB, pBBR1-kan-Ptac-RhlAB and pBBR1-kan-PrhaB-RhlAB with three different constitutive promoters (Papra、Ptac and PrhaB)were constructed by subcloning technology, and then we electro-transformated the resultant expression vectors into P.protegens Pf-5．When the flask in LB medium was fermented to 42 h, the rhamnolipid concentration of Pf-5/pBBR1-genta-RhlAB supernatant was 17.56 mg/L, with that of Pf-5/pBBR1-genta-Papra-RhlAB, Pf-5/pBBR1-kan-Ptac-RhlAB and Pf-5/pBBR1-kan-PrhaB-RhlAB supernatant being 11.135 mg/L, 441.135 mg/L and 557.764 mg/L, resulting in 0.63, 25.12 and 31.76 fold variation after promoter optimization. High performance liquid chromatography-mass spectrometry (LC-MS/MS)analysis showed that there were total 4 rhamnolipid derivatives in the Pf-5 engineering bacteria. Furthermore, the RhlAB gene expression was later examined by qRT-PCR with the result showed that the expression level of RhlAB gene after Ptac and PrhaB promoter was 2.16 and 2.77 fold higher than that of the original one. Generally, this study preliminarily realized the expression of RhlAB gene in Pf-5, and found that the constitutive promoter Ptac and PrhaB was more efficient than the original promoter which could provide a significant reference with significance for heterologous biosynthesis of rhamnolipid.