Single-molecule studies with optical techniques have become instrumental in shedding light upon the heterogeneities in molecular property distributions, which are averaged out in ensemble measurements. Gaining insights into the nature and magnitude of such heterogeneities in molecular systems, particularly when they are subject to anisotropic chemical, optical, electrical, or other perturbations, has important implications in the design of targeted detection schemes. Strategies that enable detection and manipulation of subpopulations of molecules have the potential to contribute in transformational ways to lab-on-a-chip (LOC) analytical systems and point-of-care (POC) diagnostic devices.The work in this dissertation aims to provide a novel approach for the electrochemical manipulation of redox-active fluorophores coupled with spectroscopic measurements. The design, construction, and utilization of a fluorescence correlation spectroscopy (FCS) optical system for the measurement of freely diffusing single-molecules form the basis for more advanced studies. Piezoelectric stages for nanometer spatial resolution and advanced acquisition hardware for sub-nanosecond temporal resolution are implemented. The fabrication of Au-clad zero-mode waveguide (ZMW) arrays by focused ion beam (FIB) milling is described. Simulations of the decay of the optical field within ZMWs support the basis for single-molecule experiments.Spectroelectrochemical measurements of the redox-active fluorophore, flavin mononucleotide (FMN), were performed at single-molecule concentrations in a bulk solution above an indium tin oxide (ITO) electrode and in the approximately 200 zL observation volumes of individual electrochemical ZMWs (E-ZMWs). Fluorescence analogs of standard electrochemical experiments were implemented. These are fluorescence emission under static control and chronofluorometry as analogs to amperometry, and cyclic potential sweep fluorometry as an analog to cyclic voltammetry. A long-lived semiquinone state of FMN was stabilized and observed in a ZMW. The work in this dissertation represents the first single-molecule spectroelectrochemistry investigations in an E-ZMW nanophotonic structure. Means of improving the performance of the FCS optical system and expanding its capabilities are explored. Experimental modifications, device design variations, and other possible redox-active chemical systems are discussed.