Abstract: As a green and renewable energy, bio-ethanol produced from lignocellulosic biomass is great important for China to achieve the "double carbon" strategic goal. However, the dense and complex structure composed of cellulose, hemicellulose and lignin makes it difficult for cellulase to degrade lignocellulose directly. Therefore, appropriate pretreatment was needed to disrupt the intact structure, enhance the accessibility of enzyme to cellulose, thus enhancing the ethanol production. In this study, poplar was regarded as the raw poplar, then the influence of acid-alkali combination pretreatment on hemicellulose and lignin degradation, the retention of cellulose, and subsequent simultaneous saccharification and fermentation were investigated. Subsequently, scanning electron microscopy (SEM), X-ray diffraction (XRD), and fourier transform infrared spectroscopy (FT-IR) were used to determine the effect of pretreatment on the alteration of surface morphology, composition and thermal stability. The two-step combination pretreatment included the HAc and NaOH pretreatment. The first step was conducted at (160-200 ℃) with 1% HAc catalyst. The pretreated solid obtained from HAc pretreatment was regarded as the material for the second NaOH pretreatment with different NaOH concentrations (0.3%-1.2%). The fermentation efficiency of two-step HAc-NaOH combination pretreated samples were significantly higher than that obtained from one-step HAc pretreatment. The highest ethanol concentration of 18.72 g/L was obtained from 200 ℃ HAc-0.8% NaOH pretreated substrate. The analysis of SEM, XRD and FT-IR revealed that the removal of hemicellulose and lignin during pretreatment significantly increased the cellulose content in pretreated poplar, resulting in the disruption of dense structure of poplar, which improved the accessibility of the enzyme to the cellulose and enhanced the subsequent fermentation efficiency. SEM showed that with the increase of the severity of the HAc-NaOH combination pretreatment, the structure of the poplar fiber bacame looser, the surface of the fiber was seriously broken, various cracks appeared, and the degree of fiber fracture increased. After 200 ℃ HAc-0.8% NaOH combination pretreatment, the crystallinity (CrI) was increased from 66.73% of raw poplar to 77.31%, ascribing to the removal of amorphous hemicellulose and lignin. The FT-IR analysis found that the characteristic absorption peaks of β-(1,4) -glycosidic bond (898 cm⁻¹) and-OH stretching (3340 cm⁻¹) of cellulose were observed in all samples, indicating that cellulose degraded less in the two-step HAc-NaOH combination pretreatment stage. These results were consist with the fermentation efficiency. In addition, the correlation between glucan, xylan, acid-insoluble lignin content, the removal of xylan and acid-insoluble lignin (AIL), the reserved cellulose and structural properties and ethanol yield were analyzed, suggesting that pretreatment samples with lower hemicellulose and lignin content and higher cellulose crystallinity had a higher ethanol yield.