Lignocellulosic biomass (LB), composed primarily of cellulose, hemicellulose, and lignin, is one of the most efficient renewable feedstocks for the production of sustainable bioethanol. However, its structural intricacy and chemical recalcitrant architecture present significant challenges for efficient hydrolysis and microbial fermentation. This chapter explores the complex chemistry of lignocellulosic biomass, highlighting the chemical interactions and structural characteristics that contribute to its resistance to bioconversion. The physicochemical properties of each major component have been highlighted by exploring the crystallinity of cellulose, the branching of hemicellulose, and the highly cross-linked aromatic structure of lignin. Significant focus is placed on lignin’s structural heterogeneity across varying biomass sources and pretreatment methods. In addition, the major enzymes playing a crucial role in the enzymatic hydrolysis of these three fractions have also been discussed. Their mode of action and structures of breakdown products have been provided. By integrating current understanding with molecular architecture, physicochemical properties, and transformation of lignocellulosic biomass, this chapter provides a comprehensive overview of its chemical complexity. The insights gained here aim to support the development of optimized, sustainable, and economically viable bio refinery processes.