The present work aimed to optimize a molasses and tuna-processing by-products based new economic medium for biosurfactant (BS) production by a promising strain of Aneurinibacillus migulanus. A culture medium based on a mixture of molasses and supernatants generated from tuna by-products supplemented with oligoelements solution was optimized using the mixture design methodology. Biosurfactant (BS) production and emulsification index (E24) were evaluated. Maximal BS of 2.95 g/l was obtained with a 95:5 (v:v) mixture of molasses and tuna by-product supernatant. However, higher level of E24 (62%) was recorded with medium containing the proportion 5:95 (v:v) of molasses and tuna by-product supernatant. The predicted responses from these mixture proportions were also validated experimentally. Interestingly, oligoelements supplements were not needed to prepare the culture medium. Molasses and tuna-by-product, non-conventional substrates, can be used efficiently for BS production by A. migulanus.