Deep eutectic solvents (DESs) are commonly utilized as an effective pretreatment method for biomass materials, particularly for enhancing the value of lignocellulosic biomass. However, to the best of our knowledge, the utilization of DESs for in situ pretreatment of wood samples has been seldom reported. In this study, the ability of five DESs systems to in-situ pretreatment of balsa wood samples was investigated. Among of them, the Choline Chloride-Ethylene Glycol-P-Toluenesulfonic Acid (ChCl-EG-PTSA) system showing an excellent lignin removal rate (the lignin removal of 87.34 % and cellulose retention of 86.32 %), which is significantly better than the delignification effect of the traditional acid chlorite method (lignin removal of 72,13 % and cellulose retention of 64.57 %). In addition, in the subsequent enzymatic saccharification experiments, after the wood samples were pretreated in situ by the ChCl-EG-PTSA system, the enzymatic saccharification yield of cellulose reached 75.46 %, and the enzymatic saccharification yield of xylose reached 92 %. At the same time, this study also used SEM, FTIR, XPS, TGA and other testing technologies to conduct detailed analysis and characterization of the pretreated samples. This comprehensive analysis unequivocally demonstrated the viability of the ChCl-EG-PTSA system as an environmentally sustainable and efficient approach for pretreating forest biomass. Significantly, this in-situ wood pretreatment strategy significantly lays a robust research groundwork for the development of multifunctional wood fiber composites in subsequent stages.