The depletion and environmental problems associated with fossil fuels have encouraged us to look for alternative feedstock that do not compromise food security and the environment. Sorghum is a fast-growing crop that can be harvested twice a year and produces both food and straw that can be utilized in the production of bio-based fuels. In this study, the production of bioethanol and the effects of fermentation parameters on ethanol yield are presented. A sequential pretreatment method was employed, using dilute sulfuric acid (1%) at 125 °C in the first stage and dilute sodium hydroxide (1.25 %) at 90 °C in the second stage. The residues left after the sequential pretreatment stage were hydrolyzed using acid hydrolysis. The sugar concentration of the hydrolysates was determined using the phenol sulfuric acid method, and three hydrolysates having sugar levels of 30.42 g/L, 31.79 g/L, and 32.9875 g/L were selected for fermentation. The ethanol yield was determined after 72 hours of fermentation at 30 °C with varying inoculum sizes (5%, 10%, and 15%) and pH (4.5, 5, and 5.5). With a maximum ethanol yield of 0.617 mL/g (48.742%) produced at a sugar content of 32.9875 g/L, pH of 5, and inoculum size of 15%, statistical analysis showed that all three independent parameters affected ethanol yield. According to these findings, while raising sugar content, inoculum amount, and pH all initially result in higher ethanol yields, doing so further reduces yield. So, in order to increase ethanol yield, fermentation conditions must be carefully managed while producing ethanol from sequential acidic-alkaline pretreated sorghum straw. The strategy followed by using sequential acidic-alkaline pretreatment of sorghum straw provides prospects for efficient and effective production of biofuels from alternative feedstock.