In recent decades, novel vinegar types have been developed from various fruits to meet consumer demand. However, limited research has been focused on crop-based vinegar. Therefore, this study aimed to produce vinegar from sugarcane and optimize its acidification process. Vinegar was prepared using a two-step method: yeast (Saccharomyces cerevisiae) converted carbohydrates into ethanol, followed by acetic acid bacteria (Acetobacter aceti) oxidizing ethanol to acetic acid. Physicochemical parameters, including pH, titratable acidity, total soluble solids (TSS), and alcohol content, were analyzed at 4-day intervals over 40 days under four environmental conditions (T0 Room temperature, T1 Dehydrator, T2 Incubator, and T3 Open sun). Our results showed that pH decreased in all treatments, highest in T1 Dehydrator (38.05%) and lowest in T2 Incubator (11.28%). TSS decreased most in T1 Dehydrator (80.47%) and least in T2 Incubator (66.66%). Titratable acidity increased in all treatments, with the highest increase in T1 Dehydrator (93.13%) and the lowest in T2 Incubator (85.02%). Alcohol content decreased most in T1 Dehydrator (87.5%) and least in T2 Incubator (10%). Overall, the highest changes were recorded in T1 Dehydrator, making it the most effective and rapid method for acetic acid fermentation. This study highlights a novel approach for utilizing sugarcane byproducts in value-added production.