Anodic aluminium oxide (AAO) membranes with self-ordered nanochannels have become promising candidates for applications in the aspects such as structural coloration, photonic crystals, upconversion luminescence and nanofluidic transport. Also, self-ordered AAO membranes have been extensively used for the fabrication of functional nanostructures such as nanowires, nanotubes, nanoparticles, nanorods and nanopillars. Geometries of nanochannels are crucial for the applications of AAO membranes as well as controlling growth (e.g., nucleation, direction and morphology) and in applications (e.g., optics, magnetics, thermoelectrics, biology, medicine, sensing, and energy conversion and storage) of the functional nanostructures fabricated via AAO template-based methods. However, observation of whole nanochannels with nanometer-resolution in thick AAO membranes remains a fundamental challenge, and the nanochannel geometry has not yet been sufficiently elucidated. Here, for the first time, we use depth-profiling transmission electron microscopy to reveal the truncated conical geometry of whole nanochannels of 70 μm in length. Such shape nonuniformity of the nanochannels leads to different reflectance properties of the different depths of the nanochannels along their long axis for one AAO membrane, which suggests that the nonuniformity result in some effects on applications of the nanostructures. Furthermore, we introduce a shape factor to evaluate the shape nonuniformity and demonstrate that the nonuniformity can be remarkably removed by an effective etching method based on a temperature gradient regime.