Gold nanoparticles (AuNPs) have emerged as a promising platform for a myriad of biomedical applications, including sensing, drug delivery, and antibiotics. In this thesis, I have studied and engineered the interface of AuNPs with different biological systems, demonstrating a large variety of biomedical applications by modulation of these interfaces. My research was initially focused on systematically tuning the physicochemical properties of nanoparticles to understand nano-bio interactions at the cellular level. The results demonstrate that size and surface charge of AuNP interact in an interrelated fashion to modulate nanoparticle internalization by cells, providing an engineering strategy for designing nanomaterials for drug delivery applications. Later, I engineered an environmentally responsive nanoparticle-protein interface for real time hydrogen peroxide (H2O2) sensing and monitoring of cellular oxidative stress. The responsiveness of this system demonstrates the utility of co-engineering synthetic-biological hybrid nanomaterials. Moreover, I developed gold nanoparticle-stabilized nanocapsules (NPSCs) for in vitro and in vivo gene delivery to enhance cancer therapy and immunomodulation efficiency. Finally, I demonstrated the use of gold nanoparticles to combat with pathogenic bacteria. The NP surface ligand chemistry and activity relationship reveals a new aspect to designing and constructing antimicrobial nanoparticles. In summary, the findings in this thesis highlight that systematically tuning the physicochemical properties of nanoparticles provides a powerful means to control interactions with biological systems, enabling new biological and therapeutic applications.