The ocean harbored various treasurable resources and provided great benefits to the life and health of humans. The marine oligosaccharides as a boon from nature’s desk exhibited versatile activities and applications in food, cosmetical, and medical industries. Interestingly, those oligosaccharides with different structures differed in activities and effects. Therefore, it is essential to symmetrically summarize and discuss the recent advances in source, structure, and preparation methods of marine oligosaccharides. In addition, the activities and applications mainly depended on the structures, and it is also helpful to discuss the structure-activity relationship and applications based on the literatures’ results. Herein, we summarized the lasted advances in the structure, separation, and purification of marine oligosaccharides, including the alginate oligosaccharides, carrageenan oligosaccharides, agar oligosaccharides, ulvan oligosaccharides, and fucoidan oligosaccharides with an outlook for preparation of functional oligosaccharides in food biotechnology and agriculture fields. This chapter could definitely promote the utilization of marine functional polysaccharides for food and agriculture.