To explore the antibacterial activity and structural composition of rhamnolipids produced by Pseudomonas aeruginosa A39-1 fermentation with various carbon sources, as well as to analyze the rhamnolipid biosynthesis process, the inhibitory activity of rhamnolipids against pathogens was determined using the Oxford cup assay, the congener composition of rhamnolipid samples was detected and analysed using liquid chromatography-mass spectrometry （LC-MS）, and the whole genome sequence was determined and assembled using Illumina Hiseq and PacBio. Genome sequence annotated and analysed rhamnolipid synthesis-related genes. Rhamnolipids produced by strain A39-1 by fermentation with glycerol, glucose, sunflower oil, rapeseed oil and blended oil as the sole carbon source had antibacterial activity； LC-MS detection of rhamnolipids obtained by glycerol fermentation as four monosaccharide monolipids （Rha-C_8∶2, Rha-C_9∶2, Rha-C_11∶2, Rha-C₁₀） and four monosaccharide bilipids （Rha-C_6∶1-C_6∶1, Rha-C_12∶1-C_12∶1, Rha-C₁₂-C₁₄, Rha-C_14∶2-C_14∶2）, Rha-C_11∶2 is the primary structure； The genome was anticipated to have genes for rhamnolipid synthesis related to algC, rmlA, rmlB, rmlC, rmlD, rhlA, rhlB, as well as the absence of gene rhlC encoding rhamnosyltransferase II synthesizes bi-rhamnolipids. Pseudomonas aeruginosa A39-1, a wild-type strain with a natural rhlC gene deletion, produced mono-rhamnolipids exclusively by fermentation with different carbon sources, and the mono-rhamnolipids had antibacterial activity. This study explores the antibacterial activity of mono-rhamnolipids, analyzes composition and biosynthesis pathways, and provides new ideas for the development of new green prevention and control medicines for aquatic animal diseases.