Engineered plasmonic nanostructures have emerged as powerful sensing platforms and are promoting novel applications in various fields. By providing extreme field confinement down to molecular dimensions and enhancing light-matter interaction at the nanoscale, nanoplasmonics demonstrate outstanding potential for biomolecular analysis. In addition to their extensive applications at visible and near-infrared range, such nanoplasmonic structures deliver unique possibilities for biomolecular studies with surface enhanced infrared absorption spectroscopy based on the distinct molecular vibrations in mid-infrared (mid-IR) spectral range. The mid-IR chemical fingerprints of proteins incorporate comprehensive insight into their secondary structures and thus can provide conformational information. The main goal of this thesis is to extend the applications of mid-IR nanoplasmonics in ultrasensitive detection and analysis of proteins towards enabling their in vitro characterization with innovative approaches. First, we present suspended mid-IR polarization-insensitive nanoantennas on nanopedestals for ultrasensitive vibrational spectroscopy. This system provides fully accessible near-field intensity enhancements that are realized with a new isotropic etching nanofabrication process. Our experimental results demonstrate improved sensitivity compared to the conventional antennas on substrates based on the optimized field overlap with analytes as predicted in numerical simulations. Such a mid-IR plasmonic sensor can be effectively employed in chip-based ultrasensitive detection. Next, we demonstrate for the first time the secondary structure identification of nanometer thin layers of proteins as small as 14 kDa using mid-IR plasmonic nanorods on IR transparent substrates for implementation of challenging in-solution measurements. We sensitively resolved the spectral content of plasmonically enhanced amide I fingerprint. Through its second derivative analysis, we successfully extracted major protein secondary conformations, random coil and cross β-sheet, related to aggregation of α-synuclein protein involved in Parkinson’s disease. Using an additional model protein with a native β-sheet structure, we demonstrate the sensitivity of our approach in extracting minute conformational differences. The tolerance of this new method for extracting the secondary structure signatures of thin protein layers to the resonance matching to amide I range due to any minor nanofabrication variations is an important finding for demonstrating its robustness and reproducibility. Finally, we present our biosensor integrated with a fluidic device for dynamic in situ secondary structure analysis of a protein monolayer upon external stimuli. We monitored reversible conformational changes from random coil to β-sheet in immobilized α-synuclein in real-time. Additionally, we establish the correlation between the obtained vibrational components using nanoplasmonics and that of IR transmission with polymer thin films and therefore demonstrate the reliability of our approach for mid-IR protein conformational investigations. Our platform can facilitate highly sensitive protein analysis at various conditions or in interactions with biomolecules such as lipids. These results are of great interest for multidisciplinary engineering and applied aspects of photonics and can open up a broad application area for mid-IR nanoplasmonics and devices.