Synthesis of engineered colloidal nanoparticles (NPs) with delicate surface characteristics leads to well-defined physicochemical properties and contributes to multifunctional applications. Surface engineering of colloidal NPs can improve their stability in diverse solvents by inhibiting the interparticle attractive forces, thus providing a prerequisite for further particle manipulation, fabrication of the following materials and biological applications. During the last decades, surface engineering methods for colloidal NPs have been well-developed by numerous researchers. However, accurate control of surface properties is still an important topic. The emerging DNA/protein nanotechnology offers additional possibility of surface modification of NPs and programmable particle self-assembly. Here, we first briefly review the recent progress in surface engineering of colloidal NPs, focusing on the improved stability by grafting suitable small molecules, polymers or biological macromolecules. We then present the practical strategies for nucleic acid surface encoding of NPs and subsequent programmable assembly. Various exciting applications of these unique materials are summarized with a specific focus on the cellular uptake, bio-toxicity, imaging and diagnosis of colloidal NPs in vivo. With the growing interest in colloidal NPs in nano-biological research, we expect that this review can play an instructive role in engineering the surface properties for desired applications.