Recently, the biosurfactant production has been growing up, because this substance has amphiphilic nature with high emulsifying and surface interaction. They are produced through metabolism of microorganisms and many applications are mentioned in industry (chemical, food, pharmaceutical etc.). However, the production is compromised by the use of expensive synthetic substrates. A viable alternative is resort to substrates of waste sources. This study aims to identify optimally biosurfactant production through fermentation by Bacillus subtilis using alternative substrates based waste (Glycerin from biodiesel production process, beet peel and corn steep liquor). To set the waste concentration were used design of experiments and process optimization strategies, aided by STATISTICA 7 software. It was decided use in a Central Composite Rotational Delineation 23, evaluating the responses of Emulsifying Index after 24 h(E24) and crude biosurfactant (CB). The optimum point was validated in triplicated. Experiments in scale up were performed to confirm the selected conditions. With these results, it was possible to determine the concentration of waste able to provide a good E24 and BC values.