Objective To enhance rhamnolipids production in Pseudomonas aeruginosa, an optimization strategy based on selectively blocking the metabolic bypass that competed precursors with rhamnolipids biosynthesis pathway, containing exopolysaccharide (Psl and Pel) and polyhydroxyalkanoates (PHA) synthesis pathways. Results Blocking the synthesis of Psl and PHA by genes knockout, both mutants P. aeruginosa SG increment pslAB and P. aeruginosa SG increment phaC1DC2 can grow normally in fermentation medium and increase the production of rhamnolipids by 21% and 25.3%, respectively. While blocking the synthesis of Pel, the cell growth of the mutant strain P. aeruginosa SG increment pelA was inhibited, thus its production yield of rhamnolipids was also decreased by 39.8%. In addition, simultaneously blocking the synthesis of Psl and PHA, a double mutant strain P. aeruginosa SG increment pslAB increment phaC1DC2 was constructed. Rhamnolipids production was significantly increased in strain SG increment pslAB increment phaC1DC2 by 69.7%. Conclusion Through selectively blocking metabolic bypasses, increasing the amount of glycosyl and fatty acid precursors can significantly enhance rhamnolipids production in P. aeruginosa.