Formulating a bio-based degreaser emulsion can be challenging because it requires maximizing the performance to be as good as a commercial chemical-based product without compromising the cost and effect on the environment. This study aimed to develop an eco-friendly degreaser using natural Rhamnolipid biosurfactant through response surface methodology (RSM), which was then validated and assessed compared to chemical-based commercial products. The Plackett–Burman design (PBD) and Box–Behnken design (BBD) were used to screen and optimize factors for the formulation. From the PBD's result, the amount of Rhamnolipid, with other environmentally friendly ingredients of D-limonene, sodium citrate, and calcium carbonate, were shortlisted as significant factors in the formulation. Next, the BBD optimization study revealed that only rhamnolipid and sodium citrate significantly affected the two-way interaction of the factor on the result of the surface tension and oil displacement test (ODT). In the validation study, the experimental value was very close to the rate predicted by the model for the response surface tension and ODT (24.14 mN/m and 241 mm, respectively). A comparison of the performance with commercial products proved that this formulation was comparable to that commercial product. Overall, this study revealed that the effective eco-friendly degreaser formulation was successfully produced using a minimal amount of rhamnolipid biosurfactant with a response surface methodology as a tool.