Due to its unique physicochemical properties, Pullulan is an exopolysaccharide with many applications in the food, biomedical, and pharmaceutical industries. Aiming to reduce its production cost, an interesting alternative is to consider other possibilities of raw materials, including the production of this biopolymer in a lignocellulosic biorefinery concept. Xylose is the main sugar of hemicellulosic hydrolysates obtained from different biomasses, and it is a sugar still not extensively exploited regarding its potential for pullulan production. This study aimed to evaluate the production of pullulan from sugarcane bagasse hemicellulosic hydrolysate by cultivating Aureobasidium pullulans ATCC 42023 in a bubble column reactor. The hemicellulosic hydrolysate was obtained through dilute acid treatment carried out in a stirred tank reactor before being detoxified to remove microbial growth inhibitors. The maximum concentration of 28.62 ± 1.43 g/L of pullulan was obtained after 120 h of fermentation in a bubble column reactor in batch mode. Analysis of spectroscopic properties through FTIR of the obtained pullulan revealed α-(1→6)-linked maltosyl units, similar to those of commercial samples of the biopolymer. XRD analysis showed that the prepared pullulan is amorphous, and a homogeneous morphology with a smooth surface of the pullulan was observed in SEM analysis. This study showed the potential of the production of pullulan from sugarcane bagasse hemicellulosic hydrolysate in a bubble column bioreactor, an alternative strategy for the industrial production of this biopolymer.