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Flexoelectricity refers to the electric polarisation induced by a mechanical strain gradient. By comparison with other mechanoelectric conversion approaches, the flexoelectric effect can occur in both symmetric and asymmetric dielectric materials and exhibit pronounced effects at the nanoscale. Here, we demonstrate the generation of a flexoelectric effect in a flexible nanofluidic device comprising two-dimensional (2D) graphene oxide (GO) films. Free-standing GO films are produced to construct the aligned multilayer 2D nanofluidic channels with an angstrom-scale interlayer spacing of ∼7–12 Å. Upon bending deformation, an electrical current is detected flowing between the two electrodes attached to the GO film, showing a flexoelectric coefficient up to 188 µC m−1, which is higher than that of other reported flexoelectric devices. We combine a theoretical simulation model and experimental characterisations to demonstrate that the flexoelectric effect is generated by the cross-layer transport of hydrated ions in the multilayer GO channels. The bending deformation induces a strain gradient in the GO film, which in turn drives the movement of water and ions in the charged nanofluidic channels, generating a flexoelectric output. Our nanofluidic device may be used to build self-powered sensors and actuators in the future.