AbstractRhamnolipids, simply divided into mono‐rhamnolipids and di‐rhamnolipids, their physicochemical properties are structure‐dependent. This study explored to increase the proportion of di‐rhamnolipids biosynthesized from Pseudomonas aeruginosa and evaluate the relationship between activity and di‐rhamnolipids proportions. P. aeruginosa SGrhlC was constructed by increasing the rhlC gene in P. aeruginosa SG. HPLC‐MS results indicated that SGrhlC produced more di‐rhamnolipids (62.32%) than that of the wild‐type strain SG (45.24%). Both the species and proportion of di‐rhamnolipids were increased, mainly Rha‐Rha‐C8‐C10 and Rha‐Rha‐C10‐C10. The rhamnolipids produced by SGrhlC was thermostable and salt‐tolerant. The SGrhlC rhamnolipids decreased surface tension of water to 27.0 mN/m with a critical micelle concentration (CMC) of 50 mg/L and emulsified crude oil with an emulsification index of 66.9 ± 1.5%. The SGrhlC rhamnolipids exhibited antimicrobial activity against Staphylococcus aureus and Cladosporium sp. with IC50 (half maximal inhibitory concentration) values less than 15 mg/L, and washed 73.02% ± 1.77% of oil from petroleum‐contaminated soil. Physicochemical activities of three bacterial rhamnolipids varied with their di‐rhamnolipids proportions. Results indicated that the higher proportions of di‐rhamnolipids were, the lower CMC, better surface activity and higher washing oil rate were, while the weaker emulsifying activity and lower antimicrobial activity were. The SGrhlC rhamnolipids showed better surface activity and a lower critical micelle concentration, which was superior for wetting, foaming, desorbing and dispersing. This study revealed that P. aeruginosa can be genetically regulated to biosynthesize rhamnolipids with specific structure. Perspectives of the customized biosynthesis and applications of rhamnolipids were also discussed.