A wide variety of single molecules can be identified by nanopore sensing. However, all reported nanopore sensing applications result from the same measurement configuration adapted from electrophysiology. Although urgently needed in commercial nanopore sequencing, parallel electrophysiology recording is limited in its cost and its throughput due to the introduced complexities from electronic integration. We present the first electrode-free nanopore sensing method defined as DiffusiOptoPhysiology (DOP), in which single-molecule events are monitored optically without any electrical connections. Single-molecule sensing of small molecules, macromolecules, and biomacromolecules was subsequently demonstrated. As a further extension, a fingertip-sized, multiplexed chip with single-molecule sensing capabilities has been introduced, which suggests a new concept of clinical diagnosis using disposable nanopore sensors. DOP, which is universally compatible with all types of channels and a variety of fluorescence imaging platforms, may benefit diverse areas such as nanopore sequencing, drug screening, and channel protein investigations. Natural cross-membrane transport is assisted by differe...