Pseudo-lignin derived from the condensation of carbohydrate degradation products can retard the bioconversion of lignocellulose. In this work, liquid hot water (150 to 190 °C) and 1% H2SO4 pretreatments (130 to 190 °C) were used on sugarcane bagasse holocellulose for 3 h to generate pseudo-lignin. The effects of pseudo-lignin generation on structural characteristics and bioconversion of substrates were evaluated. The results showed that the formation of pseudo-lignin increased the hydrophobicity of the substrates. After LHW pretreatments and acid pretreatments at low temperatures (<150 °C), most of the xylans were removed, yielding 2.1 to 5.4% pseudo-lignin. Increasing acid pretreatment temperature to 170 and 190 °C yielded 34.3% and 93.6% pseudo-lignin, respectively. After pretreatment, the accessibilities and bioconversions of substrates were enhanced by degradation of xylans, increasing glucose conversions and bioethanol productions of substrates from 53.2 to 85.3%, and 9.9 to 13.1 g/L, respectively. However, large amounts of pseudo-lignin were generated during acid pretreatments at 170 °C, reducing glucose conversion and bioethanol yield to 45.6% and 6.3 g/L, respectively.