The growing commitment to environmental issues, along with new legislations have made the use of synthetic surfactants become unviable by the chemical industry. Thus, the development of new technologies to produce biosurfactants increased considerably. Biosurfactants are groups of chemical compounds produced by bacteria, fungi and yeasts by biodegradation of renewable raw materials. The use of natural surfactant has become a very attractive alternative to replace synthetic surfactants due to their biodegradability, structural diversity, low toxicity and because they generate less environmental impact. Most of studies related to this topic is focused on identifying potential surfactants, in the evaluation of their properties and optimization of fermentation processes for their production by microorganisms. This study aimed to the production of biosurfactants by Bacillus subtilis using the residue from processing of pineapple enriched with glycerol as substrate, at 37 °C. Three different concentrations of glycerol, 3 %, 5 % and 10 % were used and performed in the fermentation process. It was observed that the most significant results in reducing the surface tension and hence in higher production of biosurfactants, was performed in substrate containing 3% glycerol, which was obtained 23 % of surface tension reduction during fermentation for 24 hours and increased emulsifying activity up to 62.5 %.