Most larviculture of Nile tilapia is carried out in ponds with a large presence of live food, with temperature variations throughout the day and year, and feed leftoovers due to dissipation in the water. As a consequence, the masculinization process needs to be done with a high concentration of 17α-methyltestosterone (MT) in the diet. Production in closed systems, such as biofloc technology (BFT), allows controlling the water temperature, uses a smaller volume of water and generates less effluent, being an alternative system for the masculinization process. This study evaluated the masculinization rates of Nile tilapia in the BFT, with a controlled temperature (∼28 °C), using concentrations of MT in the feed lower than those recommended for masculinization in ponds and BFT (60 mg ∙ Kg−1 of feed). Tilapia larvae (post yolk sac absorption) were raised in BFT (2 larvae ∙ L−1 or 100 larvae ∙ tank−1) and submitted to different concentrations of MT in the diet (0, 30, 40, 50 and 60 mg ∙ Kg−1 of feed), which was offered five times a day for a period of 28 days. The experimental design was entirely randomized. After the hormonal treatment, the water in the tanks was completely replaced with new water, free of hormonal residues, where the fry remained until they reached a viable size for sexing. It was also tested to maintain the original water used during the hormonal treatment period, during the post-experimental period, where 30 mg ∙ Kg−1 of feed was used. The water quality variables (temperature, pH, dissolved oxygen, settleable solids, total suspended solids, alkalinity, nitrogen compounds, salinity, phosphate and organic carbon) and growth performance variables (final body weight and length, specific growth rate, condition factor and survival) did not differ between MT concentration in the diet (p > .05). The treatment without hormone (control) presented 73.6% of males and the treatments that received hormone showed masculinization rates >99%, significantly higher than control (p < .05), but did not differ from each other (p > .05). The percentage of male individuals in the control treatment was high and this may have occurred because of the temperature of 28 °C throughout the experiment. However, this percentage of males is not considered high enough for production, which requires higher rates close to 100% as possible. The MT was not detected in the water 12 h after the last feeding with feed containing hormone. Thus, it is feasible to use a lower concentration (30 mg of MT ∙ Kg−1 of feed) in BFT. Our study has a direct impact on the global tilapia production scenario, offering a new environmentally sustainable masculinization protocol, using less water and hormones in the process and avoiding the release of hormonal residues.