Lanthanide-doped core–shell upconversion nanocrystals (UCNCs) have tremendous potential for applications in many fields, especially in bio-imaging and medical therapy. As core–shell UCNCs are mostly synthesized in organic solvents, tedious organic–aqueous phase transfer processes are usually needed for their use in bio-applications. Herein, we demonstrate the first example of one-step synthesis of highly luminescent core–shell UCNCs in the “aqueous” phase under mild conditions using innocuous reagents. A microwave-assisted approach allowed for layer-by-layer epitaxial growth of a hydrophilic NaGdF4 shell on NaYF4:Yb, Er cores. During this process, surface defects of the nanocrystals could be gradually passivated by the homogeneous shell deposition, resulting in obvious enhancement in the overall upconversion emission efficiency. In addition, the up-down conversion dual-mode luminescent NaYF4:Yb, Er@NaGdF4:Ce, Ln (Eu, Tb, Sm, Dy) nanocrystals were also synthesized to further validate the successful formation of the core–shell structure. More significantly, based on their superior solubility and stability in water solution, high upconversion efficiency and Gd-doped predominant X-ray absorption, the as-prepared NaYF4:Yb, Er@NaGdF4 core–shell UCNCs exhibited high contrast in in vitro cell imaging and in vivo X-ray computed tomography (CT) imaging, demonstrating great potential as multiplexed luminescent biolabels and CT contrast agents.