Prebiotics have long been used to modulate the gut microbiota and improve host health. Most established prebiotics are nondigestible carbohydrates, especially short-chain oligosaccharides. Recently, gluco-oligosaccharides (GlcOS) with 2-10 glucose residues and one or more O-glycosidic linkage(s) have been found to exert prebiotic potentials (not fully established prebiotics) because of their selective fermentation by beneficial gut bacteria. However, the prebiotic effects (non-digestibility, selective fermentability, and potential health effects) of GlcOS are highly variable due to their complex structure originating from different synthesis processes. The relationship between GlcOS structure and their potential prebiotic effects has not been fully understood. To date, a comprehensive summary of the knowledge of GlcOS is still missing. Therefore, this review provides an overview of GlcOS as potential prebiotics, covering their synthesis, purification, structural characterization, and prebiotic effect evaluation. First, GlcOS with different structures are introduced. Then, the enzymatic and chemical processes for GlcOS synthesis are critically reviewed, including reaction mechanisms, substrates, catalysts, the structures of resultant GlcOS, and the synthetic performance (yield and selectivity). Industrial separation techniques for GlcOS purification and structural characterization methods are discussed in detail. Finally, in vitro and in vivo studies to evaluate the non-digestibility, selective fermentability, and associated health effects of different GlcOS are extensively reviewed with a special focus on the GlcOS structure-function relationship.