Metal nanostructures with their attractive optical properties are of great interest in the field of biosensing. In this thesis, a label-free online detection of biomolecules with immobilized nanoparticles in a microfluidic chamber was realized. Two label-free sensor arrangements based on densely adsorbed (LSPR) and single nanostructures (sLSPR) were combined with a microfluidic system and compared to a commercial available system based on a gold layer (SPR). The surface sensitivity of the three sensing systems were investigated by sequential deposition of charged polyelectrolyte (PEL) layers and protein multilayers. It was even possible to detect nm thin layers online on an area smaller than 0.02µm2 on a single 80 nm spherical nanoparticle. The measurements confirmed that the LSPR signal has a non-linear dependency on the number of deposited layer in opposite to the SPR system, that the signal change is higher compared to the SPR system and that the signal depends on the size of the nanoparticle and the plasmon mode. The real-time detection of DNA-DNA binding was realized on 80 nm spherical gold nanoparticles (LSPR setup) in a microfluidic chamber without labeling. Two DNA sequences were successfully adsorbed covalently on the nanoparticles' surfaces and the subsequent hybridization was traceable within 5 min at room temperature. The additional application of non-target DNA verified the specific binding between the capture and target DNA and the binding process was repeatable by a chemical denaturation agent. Investigations concerning the buffer during biofunctionalization, the pre-treatment of the substrates with plasma and the coadsorption of mercaptohexanol resulted in an enhanced capture DNA adsorption and availability for target DNA hybridization.