One of the most versatile and safe material used in medicine is polymer-based nanomaterials. This dissertation describes the use of several formulations of polymeric nanomaterials for in vitro and in vivo optical imaging applications. In the first phase of this work, the particles assembled from diblock copolymers of poly(D,L-lactic-co-glycolic acid) and polyethylene glycol were used as a carrier for diagnostic agents. In chapter 2, the polymeric nanoparticles with a large Stokes shift of >100 nm were employed for in vivo imaging. The large Stokes shift was achieved through fluorescence resonance energy transfer (FRET) by encapsulating the donor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine) and acceptor (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine) fluorophores inside a single nanoparticle. These nanoparticles were then systematically explored to optimize the fluorophore loading and the maximum energy transfer efficiency. The animal studies further demonstrated that these nanoparticles could have far-reaching applications for in vivo imaging. In chapter 3, we further extended the study by doping the combinations of four different fluorophores, DiO, Dil, DiD, and DiR to synthesize particles that exhibited distinct emission signatures ranging from the visible to near-infrared wavelength region. This work presents first instance of nanoparticles encapsulated with four different energy transfer fluorophores inside a single particle. The optimized multicolor nanoparticles could simultaneously emit fluorescence at three different wavelengths (at 570, 669, and 779 nm) upon a single excitation (at 485 nm). Furthermore, particles with single, double, and triple emissions could be synthesized by changing the combination and doping ratio of the fluorophores. We further demonstrated that this technology could be applied to multicolor and multiplex imaging. Various physiological mechanisms are responsible for nanomaterial interaction and clearance from the blood circulation. The objective of chapter 4 was to investigate the biocompatibility, pharmacokinetics, and biodistribution of peptide-based nanofiber (NFP). In vitro studies suggested that NFP is non-toxic, hemocompatible and only showed a minimum uptake by the isolated macrophages. Upon systemic injection into mice, NFP could be delivered to the tumor in a short period of time and also eliminated rapidly by renal clearance. Overall, our results suggested that NFP is a biocompatible, safe, and effective carrier for tumoral delivery.