Abstract: Although carbon-based materials, such as graphene, metal-organic frameworks (MOFs), polymers and biomolecules, have aroused increasing scientific interest in the fields of physics, chemistry, materials science and molecular biology, their atomic-scale observation is still a challenge due to their structural instability under the electron beam. Ambiguous atomic arrangements have critically limited the fundamental understanding on these materials and their potential applications in electronics, mechanics, thermodynamics, catalysis, bioscience and medicine. Very recently, revolutionary sub-Ångström resolution achievements of transmission electron microscopy (TEM) using a low voltage, a low electron dose, or a cryogenic environment have greatly facilitated the atomic-scale structural and chemical examination of electron beam sensitive materials. In particular, the ability to image light elements atom by atom gives unprecedented insight into the structures and properties of novel carbon-based materials. In this review, the recent developments in advanced TEM combined with various imaging and spectroscopy techniques, and their use in examining graphene-based materials, MOFs, polymers, and biomacromolecules are summarized and discussed. The current challenges in materials research and trends for the future design of TEM equipment are outlined, which is expected to provide a deeper understanding of structure–performance relationships and the discovery of new carbon materials.