The recent convergence of nanomaterials and medicine has provided an expanding horizon for people to achieve encouraging advances in many biomedical applications such as cancer diagnosis and therapy. However, to realize desirable functions in the rather complex biological systems, a suitable surface coating is greatly in need for nanoparticles (NPs), regardless of the species. In this review, a recently developed surface modification strategy is highlighted--mixed-charge monolayers--with an emphasis on the nanointerfaces of inorganic NPs. Two typical mixed-charge gold NPs (AuNPs) prepared from surface modifications with different combinations of oppositely charged alkanethiols are shown as detailed examples to discuss how the mixed-charge monolayer can help NPs meet the criteria for in vitro and in vivo biomedical applications, including those critical issues like colloidal stability, nonfouling properties, and smart responses (pH-sensitivity) for tumor targeting.