Rhamnolipid (RL), the major component of biosurfactant is commonly produced via bacterial fermentation from selected carbon sources. Biosynthesis of RL is initiated by nitrogen limitation and presence of excess carbon source. RL is completely biodegradable and non-toxic. Applications of RL include the production of fine chemicals, enhancement of biodegradation, food industries and pharmaceutical products. In this study, efforts were taken to biosynthesize RL using Pseudomonas aeruginosa UMTKB-5 isolated from marine sediment. The bacterium was fed with cane sugar refinery by-product, sweetwater as sole carbon source and 5 different types of nitrogen sources. Three different of carbon to nitrogen (C/N) ratios were tested in this study. The sweetwater was first characterized for its components. Sweetwater is mainly comprised of water (79.9 wt%) and glycerol (10.3 wt%). The total sugar content is 17.4 wt% and mainly comprises of sucrose, glucose and fructose. Biosynthesis of RL was carried out in 50 mL shaken-flask cultures, incubated at 30˚C for 72 h at 200 rpm. Sulfuric acid was used to hydrolyze rhamnose groups of RL in the culture supernatant into methyl furfural. The hydrolyzed sample containing rhamnose was reacted with orcinol (1-3-dihydroxy-5-methylbenzene). The concentration of RL produced was measured spectrometrically at 421 nm. The surface tension was measured using Du Nouy Ring method. The result obtained showed that production of RL using sweetwater was in the range of 42 -50 mg/L. The cell biomass was recorded in the range of 329 -729 mg/L. The lower surface tension (47.26 mN/m) activity occurred when ammonium chloride with C/N ratio of 35 was applied. The findings of this study demonstrate the potential application of agro-industrial by-product, sweetwater as a renewable carbon feedstock for RL production via bacterial fermentation.