Natural protein assemblies have been extensively studied and exploited as biomolecular scaffolds with rich functionalities and well-defined structures. Recent advances in designing artificial protein nanostructures through self-assembling features have enabled the understanding of the basic mechanisms in protein assemblies and the construction of diverse functional biomaterials for not only biosensing and clinical diagnosis but also molecular imaging. This paper outlines the design and fabrication of novel protein nanostructures and their applications in biosensing and live cell imaging. Chapter 1 provides general research background. Chapter 2 covers the sophisticated design of the rigidity and space controllable protein assembly based on a split green fluorescence protein (GFP) system. Chapter 3 describes manufacturing a highly sensitive and specific biosensor for the detection of influenza virus by harnessing multivalency in the GFP assembly. Chapter 4 presents the development of a quantum dot (QD) based cell imaging probe for practical labeling by engineering SpyCatcher and the avidin protein.