Phospholipid-coated colloidal magnetic nanoparticles with mean magnetite core size of 9 nm are shown to be effective ion exchange media for the recovery and purification of Rhaminolipid from culture mixtures. These particles have high adsorption capacity for purification (an order of magnitude larger than the best commercially available adsorbents) and exhibit none of the diffusion resistances offered by conventional porous ion exchange media. Furthermore, purification in biological processes using colloidal magnetic nanoparticles results in saving the cost and time. In this study, production of the Rhaminolipid by Pseudomonas aeruginosa in culture media (MSM) with two types of carbon sources was carried out. Then, purification analysis was done to two types of solutions: 1. culture media without the strain 2-culture media with the strain at the different conditions of pH and ionic strength. The results of this purification method were compared to the results obtained of TLC purification method. Finally, purification of the Rhaminolipid was determined over 90% by this method.