This dissertation 1) describes the development and implementation of a single-molecule fluorescence spectroscopy technique for precise measurements, called the Anti-Brownian ELectrokinetic (ABEL) trap and 2) utilizes the ABEL trap to characterize and investigate several biological samples including designed energy transfer constructs, light-harvesting proteins found in photosynthetic bacteria, and protein-protein interactions during heat shock. Single-molecule measurements can reveal heterogeneity that is typically obscured in ensemble measurements. Conventional single-molecule techniques either measure fluorescent molecules in solution as they diffuse through a focused laser spot or measure the fluorescence from individual molecules immobilized on a surface. Freely diffusing molecules quickly move out of the observation volume due to Brownian motion resulting in short measurement times, generally on the scale of just a few milliseconds. While longer observation times can be obtained by immobilizing or tethering the molecules to the surface, it can also introduce additional heterogeneity into the measurement, thereby reducing precision. To obtain long measurements of molecules in solution without tethering them to the surface, we use an Anti-Brownian ELectrokinetic (ABEL) trap which can counteract the Brownian motion of a freely diffusing fluorescent molecule by continuously tracking its position within a region of interest and applying a feedback force to move the particle back to the center of the trapping region. Using the ABEL trap, we can observe a fluorescent molecule for tens of seconds and measure multiple photophysical parameters, such as brightness, lifetime, energy transfer efficiency, polarization, and emission spectrum. In this work, we use the ABEL trap to characterize DNA-based energy transfer constructs, termed “FRETfluors”, engineered for fluorescence multiplexing at the single-molecule level. Using just three chemical components, Cy3, Cy5, and DNA, we create dozens of FRETfluors that can be mutually distinguished from each other within one mixture using the ABEL trap. We further show the application of FRETfluors through multiplexed, wash-free detection of biomarkers at very low concentrations. Next, we used the ABEL trap to investigate a photoprotective mechanism found in cyanobacteria which involves the interaction of the light-harvesting complex, called the phycobilisome, and the Orange Carotenoid Protein (OCP). We compare this photoprotective mechanism among two species of cyanobacteria containing phycobilisomes with different macromolecular architectures. We find that the OCP binds at distinct and specific sites in each type of phycobilisome while providing nearly identical quenching strength in both cases. Finally, we explore the possible applications for two-color laser excitation within the ABEL trap. Using Pulsed Interleaved Excitation (PIE), we obtain accurate FRET measurements, increase the number of mutually distinguishable FRETfluors, and discover stoichiometry of proteins that interact when cells undergo stress, such as heat shock. This dissertation demonstrates the wide utility of the ABEL trap platform for precise single-molecule fluorescence measurements to explore complex biological systems and gain new insights into molecular interactions and photophysical properties at the single-molecule level.