Graphene oxide is a popular material because of not only its cost–efficient production but also its unique properties. However, after long-term storage, several changes occur in the structure of graphene oxide obtained using the modified Hummers’ method, affecting its stability and physicochemical properties. Despite the obvious problem, there is only a limited number of works devoted to ageing factors, their impact on the properties and structure of graphene oxide itself, and operational modes of materials and devices based on graphene oxide. Therefore, to facilitate the widespread industrial application of graphene oxide, it is crucial to establish standardized protocols for storage and quality assessment of graphene oxide. In this paper, the prepared samples were extensively characterized using various techniques, including transmission electron microscopy, X-ray photoelectron spectroscopy, UV–Vis and fluorescence spectroscopy techniques. Among the various analytical methods, comprehensive spectroscopic analysis of the absorption and fluorescence properties has proven to be the most sensitive to sample ageing. We investigated the change in optical and physicochemical properties of graphene oxide during its long-term storage under various conditions. It was found that graphene oxide precipitated during storage, and the O/C ratio in graphene oxide decreased slightly from 0.49 to 0.47. Still, the type of oxygen-containing functional groups did not change, which might indicate possible intra-structural changes. Using transmission electron microscopy, molecular dynamic and density functional theory simulation, we showed that the rearrangement of functional groups on the surface leads to the formation of large sp2 domains that interact with each other, leading to aggregation of the graphene oxide.