Rhamnolipid biosurfactants were extracted and purified from the fermentation broth of Pseudomonas aeruginosa. The composition of rhamnolipid extract was analyzed by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS). The effect of pHvalue on its surface activity was studied by determining its critical micellization concentration (CMC). The effect of pH value on the microstructure of the rhamnolipid/n-heptane/borate microemulsion system was evaluated by considering particle sizes and zeta potential of microdroplets. Results showed that under weak alkaline conditions (pH 7.5-9.0), pH had significant effect on both the surface activity of rhamnolipid and the microemulision microstructures. At a pH lower than 9.0, the CMC decreased as pH increased and reached a minimum at pH 9.0. At a pH higher than 9.0, the CMC increased gradually as pH increased. This was as a result of a joint effect of hydrogen bonding and electrostatic repulsion between polar head groups. Both size and absolute value of zeta potential of microdroplets tended to increase as pH increased except at pH 9.0. By adding a small quantity of sodium dodecyl sulfate (SDS) or n-butanol to the microemulsion system, the particle size increased significantly.