The immobilization of S. passalidarum in calcium alginate beads for second-generation ethanol production (E2G) was evaluated in a medium that simulated a hemicellulosic hydrolysate of sugarcane bagasse pretreated with diluted sulfuric acid in terms of sugars composition. Three sets of sequential batch fermentations (SBF) were carried out with free cells, or immobilized cells in high (HSC) and moderate (MSC) initial sugar concentration (120 and 70 g/L, respectively). SBF were characterized by five consecutive batches performed in shaker, at 30 °C and 110 rpm. Better results were observed for the SBF with immobilized cells in MSC medium when compared to HSC (Y’P/S of 0.27 ± 0.02 and 0.19 ± 0.03 g/g, respectively), in the second batch cycle. The value for YP/S in MSC was similar to the obtained with free cells (0.30 ± 0.02 to 0.33 ± 0.02 g/g). However, QP was lower for MSC, reaching 0.81 ± 0.04 g/L.h in in the second batch, while for free cells the QP varied from 1.06 ± 0.02 to 1.16 ± 0.22 g/L.h. A technique for determining the concentration of immobilized cells in the alginate beads was applied, which made it possible to determine the specific rates for the SBF performed. According to the results obtained, it was possible to demonstrate that S. passalidarum can be immobilized in calcium alginate and reuse through SBF, with performance similar to free cells, which can be a good strategy for fermentation of hemicellulosic hydrolysates.