A layer-by-layer (LbL) polyelectrolyte deposition strategy is used to prepare multifunctional nanoparticles (MFNPs) with multimodal imaging capabilities. Alternating treatment of hybrid silica nanoparticles (NP0) containing a luminescent [Ru(bpy)3]Cl2 core and anionic monolayer coating of the Gd-(siloxylpropyl)diethylenetriamine tetraacetate (Gd-DTTA) complex with cationic Gd(III)-DOTA oligomer 1 and anionic poly(styrene sulfonate) (PSS) led to the deposition of multilayers of 1 and PSS via electrostatic interactions. This LbL deposition technique offers a superb strategy for the assembly of hybrid nanoparticles with imbedded luminophores and very high MR relaxivities. The PSS-terminated multilayered nanoparticles can be noncovalently functionalized with targeting peptides that carry positive charges under physiological conditions via electrostatic interactions to lead to cancer-specific MFNPs for optical and MR imaging of HT-29 human colon cancer cells. The generality of this approach should allow the design of imaging and/or therapeutic MFNPs that can specifically target a wide range of diseased cells.