Ethanol was produced from alfalfa stems with ambient-temperature sulfuric acid pretreatment, ensiling, and washing. The cellulose and hemicellulose contents of ensiled alfalfa stems decreased, while the ash and water-soluble carbohydrates contents increased with increasing sulfuric acid during pretreatment. The air-dry process had a negative effect on ethanol production. The ambient-temperature sulfuric acid pretreatment and washing greatly improved ethanol production from alfalfa stems. We theorize that sulfuric acid at ambient temperature partially degraded hemicellulose and ash, and altered cell wall structure during long-term ensiling, and washing removed soluble ash in substrates that could inhibit the simultaneous saccharification and fermentation.