The utilization of lignocellulosic materials for second-generation ethanol production via enzymatic catalysts is primarily hindered by enzyme cost. Enzymatic immobilization emerges as a viable solution, enabling enzyme reuse. This study investigated the immobilization of an enzymatic extract obtained from Moniliophthora perniciosa fermentation in calcium alginate spheres using the direct trapping method. Initial tests assessed β-glucosidase activity, showing that a higher concentration of calcium chloride (1 M) alongside larger diameter spheres yielded improved results. The immobilized enzyme was reused for up to 17 cycles without significant loss of activity. The percentage of reducing sugars after 48-h hydrolysis with the supplemented enzymatic extract was 226%, doubling the value achieved with only the free enzymatic extract. The immobilized enzyme retained 50% of its initial activity after 1 h at 80 °C, demonstrating higher activity at pH 6 and 60 °C. These findings suggest that this immobilization technique is simple, economically viable, and effective for the hydrolysis of pretreated sugarcane bagasse.