Abstract: In this study, Yunnan arabica coffee beans were selected as the research object, and the extraction process of coffee melanoidins were optimized. Filter membrane fractionation of 100, 50 and 30 kDa were used to characterize the structure of coffee melanoidins of different molecular weight segments based on molecular morphology and spectroscopy characteristics. The results showed that the optimal extraction conditions were as follows: Solid-liquid ratio 1:16 (g/mL), extraction temperature 80 ℃, extraction time 60 min. The results of gel permeation chromatography showed that the coffee melanoidins tight spherical structure in the solution, and the molecular weight were mainly 4.56×10⁴ and 2.94×10⁴ Da, accounting for 72.5% and 27.5%. X-ray diffraction showed that the coffee melanoidins were amorphous and had weak crystallization. The analysis of micromorphological characteristics showed that the coffee melanoidins without fractionation treatment were irregularly agglomerated spherical with rough surface and pores. After fractionation treatment, the polymerization structure disappeared and showed irregular flake and long strip shape. The degree of black-brown and the content of basic components decreased with the decrease of molecular weight. The UV-Vis spectra showed that different molecular weight components had weak absorption peaks at 285 and 320 nm. Fourier infrared spectroscopy showed that there might be carboxyl, hydroxyl, amino and methyl groups. The 3D-EEMs had λex=400 nm, λem=480~490 nm, indicated that coffee melanoidins concentrate had aromatic ring, heterocycle and rigid structure. Through the optimization of extraction, fractionation and structure characterization of coffee melanoidins, it would provide certain data support for further research on the formation, transformation and basic structure of Yunnan arabica coffee melanoidins.