Engineered nanomaterials have been increasingly utilized in industry for various consumer products, environmental treatments, energy storage, and biomedical applications. Meanwhile, it has been established that certain nanomaterials can be toxic to biological cells from extensive experimental and theoretical studies. Despite that the exact molecular mechanisms of this nanomaterial toxicity are still not well understood, it is ubiquitous that their interactions with cell membranes, through either endocytosis or penetration (and thus potential lysis), act as the first step toward the inflammation or even the death of a cell. To facilitate the study of nanomaterial-membrane interactions, here we demonstrate a nanopore-based single-molecule approach that can be applied to monitor a specific nanomaterial-membrane interaction in real time. Combined with molecular dynamics and experimental approaches, we show how an ionic current can be used to detect membrane damage by a graphene nanosheet and illustrate the underlying molecular mechanism. More generally, we expect that measured transmembrane ionic currents (both DC and AC) can signify many particle-induced membrane modifications, such as hole formation, particle adsorption, and protein insertion.