The effect of oil-to-glucose ratio was studied for biosurfactant production by using sequencing batch reactors (SBRs) and a bacterial strain which was isolated from a petroleum-contaminated soil in Thailand, Pseudomonas aeruginosa SP4. The studied SBRs were operated at a constant temperature of 37°C under aseptic conditions with a 1,500 ml working volume, 500 ml feeding volume, and 500 ml decanting volume. Palm oil and a mineral medium were used as the carbon source and the nutrient source, respectively. Glucose (another type of carbon source) was added to the mineral medium for the enhancement of microbial growth which, in tern, increased the biosurfactant production. From the results, the optimum oil-to-glucose for a maximum yield of biosurfactant production was 40/1. It provided a surface tension reduction of 58.5% and a surface tension of 29.9 mN/m, corresponding to a highest COD and oil removal of 85.1% and 77.7%, respectively. Under the optimum conditions, the biosurfactant was produced eruptly after 6 h of the aeration as indicated by the sharp drop of the surface tension and remaining constant at around 28 to 31 mN/m. The critical micelle dilution (CMD) method showed that the biosurfactant concentration at the 42 to 48 h aeration time was 1.11 times the CMC.