Surface plasmon resonance sensors of the nanohole array type provide a promising platform for label-free biosensing on surfaces. For their extensive use, an efficient fabrication procedure to make nanoscale features on metallic films is required. We develop a simple and robust template-transfer approach to structure periodic nanohole arrays in optically thick Au films on poly(dimethylsiloxane) substrates. This technique significantly simplifies the process of sensor fabrication and reduces the cost of the device. A spectral analysis approach is also developed for improving the sensor performance. The sensitivity of the resulting sensor to refractive index change is 522 nm/RIU (refractive index unit) and the resolution is improved to 2 × 10(-5) RIU, which are among the best reported values for localized surface plasmon resonance sensors. We also demonstrate the limit of detection of this sensor for cardiac troponin-I.