Abstract Food supplements have been increasingly investigated. Probiotics have several benefits for human and animalhealth and selenium (Se) is widely recommended against oxidative stress. In this context, the aim of this study was to develop a low-cost bioprocess to produce a functional food product comprising both probiotic and Se accumulation. Yeastcells of Saccharomyces boulardii CCT 4308 were cultivated using sugarcanemolasses as substrate. Optimization studies were performed to evaluate the best medium composition for biomassproduction and Se-accumulation in batch and fed-batch systems. Optimized conditions were defined with a medium composed of 150 g L-1 sugarcanemolasses and 12 g L-1 yeast extract, with feeding of 100 g L-1 sugarcanemolasses and 100 μg mL-1 of Se incorporation after 4 h and 10 h of fermentation, respectively, during 48 h in STR (stirred tank reactor). Best biomassproduction reached 14.52 g L-1 with 3.20 mg Se g-1 biomass at 12 h. Process optimization led to 4.82-fold increase in biomassproduction compared to initial condition. A final Se-enriched S. boulardii CCT 4308 biomass was obtained, which is comparable to commercial products. An alternative probioticyeastbiomass was efficiently produced as a new food-form of Se supplement in a sustainable process using an inexpensive agro-industrial residue.