Abstract Rhamnolipid is the simplest class of biosurfactants with a well defined structure. The main obstacle in commercialization of biosurfactant especially rhamnolipids is the higher cost of the production. The production cost can be reduced by using economical carbon sources. Glycerol residue is available from glycerin distillation plant at low cost. It can be used as carbon source for rhamnolipid production using Pseudomonas aeruginosa (ATCC 10145). The optimum concentration of glycerol residue was 5% weight by volume (w/v) yielding maximum rhamnolipids at 2.50 g/L in the broth. From batch monitoring of polyglycerol and monoglycerol consumption, it appeared that polyglycerols were first hydrolyzed to monoglycerol in the broth itself and consumed as carbon source. The product had components similar to rhamnolipids synthesized on pure glycerol under the same conditions. This indicated the potential of glycerol residue as economic substrate for production of rhamnolipids.