Rhamnolipids are promising biosurfactants with diverse applications, but their efficient production from glucose remains challenging. In this study, a glucose-based fermentation process was developed using Pseudomonas aeruginosa YM4 through sequential evaluation of nitrogen source, pH, C/N ratio, glucose concentration, and Fe²⁺ supplementation in a 5 L bioreactor. A mixed NH₄⁺/NO₃⁻ nitrogen source resulted in higher rhamnolipid production than either NH₄⁺ or NO₃⁻ alone. Maintaining the culture pH at 7.2 prevented excessive acidification and promoted glucose consumption and rhamnolipid production. The highest rhamnolipid titer (29.7 g/L) was achieved using 100 g/L glucose, a C/N ratio of 35, and 10.8 μM Fe²⁺. Fermentation kinetics indicated that biomass accumulated rapidly during the early fermentation stage, whereas rhamnolipid production continued after cell growth had slowed. Under the selected conditions, YM4 produced a rhamnolipid mixture containing 73.8% di-rhamnolipids and showed substantially higher rhamnolipid production than three additional P. aeruginosa strains cultivated under identical conditions. This study developed a glucose-based fermentation process for producing di-rhamnolipid-rich products and demonstrated the efficient rhamnolipid-producing capability of P. aeruginosa YM4 under the evaluated conditions.