Glycosylation modifications, extensively present on the surfaces of eukaryotic proteins as a type of post-translational modification, hold significant physiological and pathological implications. The microscopic heterogeneity of natural glycoproteins has led to the emergence of the chemical synthesis of homogeneous glycoproteins with defined structures as a crucial frontier in exploring the structure-function relationships of glycosylation modifications. With the flourishing development of protein synthesis and glycoengineering technologies, various protein ligation and polysaccharide synthesis strategies have been developed, enabling the preparation of glycoproteins containing hundreds of amino acid residues. The development of glycoprotein synthesis strategies primarily revolves around chemical and enzymatic approaches for glycosidic bond formation, leading to effective synthesis schemes such as Lansbury’s aspartic acid acylation, chemical strategies based on glycosyl amino acid building blocks, and glycan remodeling strategies using endoglycosidases and glycosyltransferases. This review will discuss the chemical and enzymatic construction of glycosidic bonds, examining existing strategies for the total synthesis of glycoproteins and semi-synthetic approaches that combine with biological expression methods. It will introduce these strategies’ achievements in synthesizing complex homogeneous glycoproteins with different types of glycosylation modifications, such as those with multiple complex N-glycosylation modifications like HSV gD and those containing long hydrophobic segments like IL-2. Additionally, this review will highlight breakthroughs in understanding the structure-function relationships of glycosylation modifications in various physiological processes through these synthetic complex glycoproteins, including the relationship between glycan chain length and immunogenicity in antigenic glycoproteins, and the mechanisms by which O-GlcNAc regulates synaptic function in neurons. Finally, it will summarize the progress made in glycosidic bond construction, purification strategies, and protein solubility, and point out that further optimization of selectivity and synthetic yield remains a pressing issue in the field of glycoprotein synthesis. The wide application of glycoprotein synthesis technology in developing immunotherapies and understanding the molecular mechanisms of various diseases expands the development directions of synthetic science in the field of life and health, from understanding principles to developing products.