Abstract: Acetic acid(AC) hydrolysis and the second-step pretreatment were used to produce xylooligosaccharides(XOS) and enzymatic hydrolysate from poplar. The hydrolysate was used as carbon resource to produce Bacillus subtilis by fermentation. After the poplar was pretreated by 5% AC at 170℃ for 30 min, the XOS yield of the pretreatment liquor was 55.8%. In the second-step pretreatment, the order of improvement on enzymatic hydrolysis of AC-pretreated poplar was showed as follows: hydrogen peroxide-acetic acid(HPAC), sodium hydroxide, sodium hydroxide-hydrogen peroxide, γ-valerolactone(GVL), ammonia pretreatment. The AC-pretreated poplar after HPAC pretreatment had the highest digestiability and the glucose yield of the hydrolysate was 72.4%. Specially, Tween 80 could further improve the glucose yield of AC-HPAC-pretreated poplar to 93.8%. The enzymatic hydrolysate obtained from the two-step pretreated poplar was used to produce B. subtilis and the highest viable count of the fermentation liquor could reach 2.1×10⁹ CFU/mL. After fermentation for 11.5 h, the utilizations of glucose and xylose were 95.3% and 25.0%, respectively. The results in this work could guide for the co-production of XOS and B. subtilis from poplar, and provide a reference for the high value-added conversion of poplar.