As the most dynamic and diverse macromolecules in our body, proteins perform a vast array of function, such as catalyzing metabolic reactions, forming receptors and channels for transportation, responding to stimuli and etc. In this context, tremendous opportunities are provided in harnessing proteins for therapeutic and diagnostic purposes. However, there are still challenges in the development and delivery of protein therapeutics. For instance, the vulnerable nature of proteins with poor stability lead to alternation of protein architects during delivery process which hindered its application. In addition, the low permeability of native protein though biological barriers (e.g., cell membrane, the mononuclear phagocyte system, blood brain barrier, etc.) prevents the successful delivery and efficient response of protein therapeutics. Therefore, development of novel protein delivery platforms, which can stabilize proteins and overcome biological barriers, will broaden the utility of protein therapeutics. In this dissertation, novel platforms for protein delivery have been developed based on the protein nanocapsule technology, which is achieved by encapsulating the protein molecules with a thin polymer network via in situ polymerization. Such protein delivery platform can significantly improve the protein stability as well as endow various surface properties (e.g., cationic charge, stealth surface, specific targeting capability, etc.) to overcome different biological barriers. Based on this technology, we enable to rationally design and synthesize nanocarriers, understand and precisely control behaviors during transportation process through the biological barriers. This dissertation can be outlined with the following three topics: 1) Fully understand and precisely control the kinetics of intracellular protein delivery. In this part, FLuc nanocapsules, which can mimic current strategies for intracellular delivery, was employed as a probe to real-time quantify the internalization process. By realizing precisely spatiotemporal control over distribution and functions, this platform provides a simple and efficient approach for optimization of dosimetry, characterization of therapeutic efficiency and screening of novel medicine. 2) Overcome the mononuclear phagocyte system to deliver protein therapeutics with prolonged circulation time and reduced immunogenicity. In this work, another FLuc nanocapsule with stealth surface was developed. The probe improves FLuc stability, reduces the immune clearance, prolongs the circulation time and present a high contrast imaging of the tumor. This method provides an effective and safe route for tumor diagnosis. Overall, this dissertation established various novel strategies toward better protein delivery platforms for overcoming biological barriers, which broaden the application of protein therapeutics.