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This letter addresses the characteristic of ion-selective field-effect transistors (ISFETs) with periodic-groove channels. The channels with the groove width/space about 1:1, 200/200 nm, were fabricated by ultraviolet nanoimprint lithography. The proposed ISFETs exhibited higher sensitivity, lower hysteresis, and lower drift characteristics than single channel. The nanoscale corners of periodic grooves can improve the surface potential response to the ion concentration. With this approach, high-performance ISFETs can be fabricated for future biosensor applications.