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We present radio-frequency (RF) heating-assisted nanoimprint lithography (NIL) for the rapid fabrication of nanostructured biochips. The chips were spin-coated using a RF-sensitive polymer film, namely polyethylene terephthalate glycol-modified (PETG). The RF heating process takes advantage of rapid temperature increases and cooling for NIL. Using a patterned nickel film as the mold, various nanostructures, such as nanowire, nanorod, and nanogrid arrays, were successfully fabricated within several seconds. For surface plasmon biosensing chips, the silver-coated nanowire arrays achieved a linewidth of 6.01 nm and wavelength sensitivity of 550 nm per refractive index unit. The functionality of the sensor was verified by observing the label-free antigen–antibody interactions.