Biosurfactant produced by Pseudomonas aeruginosa UKMP14T was optimized by growing the isolate in mineral salt medium (MSM) supplemented with 1% (v/v) glycerol and 1.3 g/L ammonium sulphate with C/N ratio of 14:1. The culture medium was incubated at 37°C, with an agitation speed of 150 rpm for 7 days. P. aeruginosa UKMP14T produced biosurfactant at 0.8 g/L after 7 days incubation. Anthrone assay proved biosurfactant was glycolipid. The biosurfactant was characterized by scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDX) in addition to Fourier transform infra-red (FT-IR), nuclear magnetic resonance (NMR) and electrospray ionization-mass spectrometry (ESI-MS). The SEM-EDX analysis indicated the presence of carbon and oxygen elements by 78% and 22% (atomic %), respectively. FT-IR absorption spectra indicated the functional groups of rhamnolipid were located at 3308. 46, 2922.91, 2857.09 and 1730.96 cm -1 . ESI-MS/MS analyses identified P. aeruginosa UKMP14T produced rhamnolipid with two fatty acids-nine congeners, L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C 10 -C 10 ) (m/z 649) which formed the main compound. The critical micelle concentration (CMC) of this rhamnolipid was established at 30 mg/L (32 dynes/cm). The characteristics of biosurfactant produced by P. aeruginosa UKMP14T indicated it has a high potential in industrial dan bioremediation application. Keywords: biosurfactant, Pseudomonas aeruginosa, rhamnolipid, spectroscopic analysis, CMC value AbstrakBiosurfaktan dihasilkan oleh Pseudomonas aeruginosa UKMP14T dioptimumkan dalam medium garam mineral (MSM) yang ditambahkan dengan 1% (i/i) gliserol dan 1.3 g/L ammonium sulfat dengan nisbah C/N iaitu 14:1. Medium kultur ini dieram pada 37°C, dengan kelajuan goncangan pada 150 psm selama 7 hari. P. aeruginosa UKMP14T menghasilkan biosurfaktan sebanyak 0.8 g/L selepas 7 hari pengeraman. Asai antron membuktikan biosurfaktan terhasil adalah glikolipid. Biosurfaktan tersebut dicirikan oleh mikroskop pengimbasan elektron-spektroskopi tenaga serakan sinaran-X (SEM-EDX), dengan penambahan Infra-merah transformasi Fourier (FT-IR), resonan magnetik nuklear (NMR) daknn spektrometri jisim-pengionan elektrosemburan (ESI-MS). Analisis SEM-EDX menunjukkan kehadiran unsur karbon dan oksigen masing-masing sebanyak 78% dan 22% (% atomik). Spektrum penyerapan FT-IR menunjukkan kumpulan berfungsi ramnolipid pada 3308.46, 2922.91, 2857.09 dan 1730.96 cm -1 . Analisis ESI-MS/MS mengenalpasti P. aeruginosa UKMP14T menghasilkan ramnolipid dengan sembilan kongener-dua asid lemak, L-ramnosil-L-ramnosil-β-hidroksidekanoil-β-hidroksidekanoat (Rha-Rha-C 10 -C 10 ) (m/z 649) sebagai sebatian utama. Kepekatan misel kritikal (CMC) ramnolipid ini dikesan pada 30 mg/L (32 dynes/cm). Ciri-ciri biosurfaktan yang dihasilkan oleh P. aeruginosa UKMP14T menunjukkan ia mempunyai potensi tinggi untuk di aplikasi dalam industri dan bioremediasi.