Efficient medical care fundamentally relies on the ability to provide a timely and accurate diagnosis. Thanks to advances in biomedical research, specific molecules called diagnostic molecular biomarkers have been discovered in the human body that help indicate diseases in highly specific ways. The small size and low concentration of many of these molecules pose a serious challenge to detecting them from the rich content of human blood and other liquid biopsies. Medical laboratories use large and complex tools to enable sensitive and robust quantification of such biomarkers. However, these approaches are time-consuming, require expensive equipment and delay the doctors' decision-making. Therefore, compact, cost-effective and rapid technologies that enable testing of biological fluids to identify low-abundance biomarkers directly at the patient’s bedside are critically needed to assist the modern healthcare. This doctoral thesis presents a novel biosensor that enables highly sensitive, accurate and rapid detection of disease biomarkers in a low-cost and portable device directly from patient blood serum. The first major and original contribution is on the introduction of an innovative sensing principle that uses sub-wavelength gold nanoparticles and large area periodic gold nanohole arrays. The nanohole arrays consist of millions of nano-perforations in a thin gold metal film on a glass substrate and enable a plasmonic phenomenon called extraordinary optical transmission. The interactions between nanoparticles and nanoholes are imaged in a spectrometer-free set-up and enable the detection of individual molecule binding in complex samples. Unlike conventional plasmonic sensing approaches that rely on spectral shifts of plasmonic resonances, our method exploits intensity modulations caused by individual nanoparticles on nanohole arrays. Therefore, the technology overcomes classical plasmonic detection limits imposed by refractive index sensitivity. The work shows that the biosensor achieves highly sensitive detection, meeting clinically relevant concentrations, and can provide a powerful platform for biomarkers testing. The second major and original contribution includes the integration of the novel plasmonic sensor technology into a portable point-of-care (POC) device. It is deployed in a hospital and validated with a wide range of patient samples with inflammatory conditions. The device enables ultra-sensitive detection of two sepsis-related biomarkers, procalcitonin, and C-reactive protein. The tests with biobank patient samples revealed that the novel POC device provides diagnostic performance equivalent to gold standard laboratory immunoassays. Moreover, identification of biomarker levels can be performed in under 15 minutes on-site, providing critical advantage compared to laboratory testing. The results of this thesis build upon a broad interdisciplinary knowledge ranging from engineering (including plasmonics, imaging, nanofabrication, and device integration) to chemistry, biology, and medical diagnostics. The plasmonic sensing principle introduced in this work offers a promising strategy for the development of many new biosensing applications, while the developed point-of-care biosensor has the potential to provide a rapid and accurate tool to assist the diagnosis and management of diseases in various settings, improving the quality of medical care for more people.