<p>The production of second-generation ethanol from tobacco stem has the potential to boost sustainability in the tobacco industry; however, the lack of xylolytic microorganisms in many studies impairs the conversion process of stem hydrolysate into bioethanol. The utilization of <i>Spathaspora passalidarum</i> yeast in hydrolysate fermentation represents a significant advancement in valorizing this residue in biorefineries, as the yeast can utilize both xylose and glucose for ethanol production, along with by-products such as xylitol and glycerol. The study investigated second-generation ethanol production from tobacco stem through a multi-step process involving alkaline pretreatment followed by enzymatic hydrolysis and separate fermentation by <i>S. passalidarum</i> yeast. The solids obtained from pretreatment showed low acetyl and extractive content, which can act as inhibitors of enzymatic hydrolysis and fermentation. Enzymatic hydrolysis was performed in two steps due to the low yield of the first step. The global conversion yield (first step + second step) into glucose and xylose was 47% and 58%, respectively. Pretreatment and enzymatic hydrolysis changed the morphology of cellulose structure of tobacco stem according to SEM and XRD observations. Through <i>S. passalidarum</i> fermentation was possible to attain 11&#xa0;g.L⁻¹ ethanol, yield of 76.32%, productivity of 0.74&#xa0;g.L⁻¹.h⁻¹, and 0.13&#xa0;g.L⁻¹ xylitol. The mass balance of the entire process was presented considering 1 ton of tobacco stem as input, resulting in 53 L ethanol.</p> Graphical Abstract <p></p>