Abstract: Glycosylation is one of the most common protein post-translational modifications (PTMs) and plays a critical role in regulating various biological functions by influencing protein folding, trafficking, and subcellular localization. Aberrant glycosylation patterns are closely associated with the onset and progression of numerous health conditions, including cancer, inflammation, autoimmune disorders, and other diseases. Glycoproteomics, as a key field dedicated to studying protein glycosylation, has demonstrated increasingly significant value in the discovery of disease biomarkers and the investigation of underlying pathogenic mechanisms. Human body fluids serve as a crucial “window” into disease information, with their glycoproteomes harboring rich pathophysiological insights. Mass spectrometry (MS) is a powerful tool for deciphering the glycoproteome of body fluids. In this review, a concise overview of the types and biological processes of glycosylation was provided, followed by an introduction to MS-based glycoproteomic workflows. Recent advances in MS-based glycoproteomic studies involving various human body fluids, such as blood, urine, cerebrospinal fluid, tears, seminal plasma, milk, and saliva, were summarized with emphasis on current research regarding the types, abundance, and structural features of glycoproteins and glycans identified in these fluids. Furthermore, trends in glycosylation alterations under disease conditions and potential glycoprotein-or glycan-based biomarkers and therapeutic targets that have been discovered were highlighted. This review aims to offer a comprehensive perspective and reference for understanding the role of fluid-based glycoproteomics in disease and facilitating its clinical translation.