Surface plasmon resonance imaging (SPRi) is capable of real-time, label-free, and high-throughput detection for many sensing applications. The conventional prism-based SPRi system is operated with intensity interrogation. Because the intensity-based sensors are easily disturbed by the environmental conditions such as light absorption of the analytes and source intensity noise, the intensity variations must be corrected. Besides, the sensitivity of intensity-based SPRi is moderate. To address the issues, we propose a low-cost, portable nanostructure-based SPRi platform, composed of a commercial transmission scanner, aluminum (AO-based nanostructure chips, and self-referencing two-color analysis, for high-throughput sensing applications, such as the thickness of the dielectric film, bulk refractive index of the solution, and biomolecular interactions. Al nanoslit and capped nanoslit arrays are fabricated using nanoimprinting technology, and their bulk and thickness sensitivities are studied. The results show that the refractive index and thickness sensitivities are 3260%/RIU and 16.2%/nm, respectively. The detectable refractive index resolution and surface thickness are 4.3 X 10(-5) RIU and 0.053 nm, respectively. The protein-protein interactions between bovine serum albumin (BSA) and anti BSA proteins are conducted, and the minimum concentration of anti-BSA proteins, 10 pg/mL, is detectable. In addition, the high-throughput detection of the surface thickness is demonstrated, and 96 sensing arrays are quickly monitored. Such a portable, low-cost, and high-throughput sensing platform can benefit various multiplex sensing applications.