Directed excitation energy transport at the nanoscale in supramolecular assemblies is of great importance for the development of new organic optoelectronic devices and next-generation solar cells. It is well known that the highly symmetric arrangement of the chlorophyll pigments in the natural light-harvesting systems promotes efficient directional excitation energy flow to the reaction center. This highly directed energy transport in such natural light-harvesting systems has been a great inspiration for their artificial counterparts, nano-antenna systems. For instance, organic dyes, polymer conjugates, organic crystalline films, and various types of inorganic nanoparticles are among the best candidates due to their high exciton mobility at the nanoscale level. However, the precise control of chromophore spacing and organization, taking into account the inhomogeneity and defects of such nanomaterials during self-assembly preparation, remains a challenge for these artificial light-harvesting systems. A comprehensive understanding and characterization of the structural insight and energy transfer dynamics of such molecular assemblies is required to control/alter their functionality for the development of new optoelectronic devices. For their practical application, it is also necessary to immobilize these nanostructures on solid substrates without destroying their supramolecular structures, optical and photophysical properties. Recently, precise fluorescence microscopy imaging has been implemented as a novel technique in various nanostructured materials for in situ and real time visualization of their dynamic behavior, visual characterization of defects in metallic organic 3D frameworks and liquid crystals, inhomogeneity in inorganic hybrid crystalline nanostructures, conformational analysis and molecular weight determination of polymers and characterization of polymerization progress and micro-dispersion in organic-inorganic composites. Therefore, microscopy imaging can be used as a simple tool to understand and visualize the light-harvesting phenomena of supramolecular nanostructures probed by energy transfer. Polarization experiments, localizing both position and orientation of the chromophore units that are present in such assemblies, can well characterize the influence of the anisotropic shape/structure of the supramolecular assembly on its light-harvesting property. We have reported the supramolecular assembly of a chiral bichromophoric naphthalenediimide (molecule (R)-1NDI in Scheme 1, left) into nanofibers, which showed absolute enantioselective recognition of a chiral perylenediimide bichromophoric derivative (molecule (S)-2PDI in Scheme 1, right) with opposite handedness by host-guest interaction in methylcyclohexane (MCH) rich solution. This chiral recognition of nanofibers towards the (S)-2PDI guest molecule was evaluated by Förster resonance energy transfer (FRET),where the fast and efficient exciton migration among the (R)-1NDI chromophore units along the nanofibers efficiently transport the excitation energy to the bound (S)-2PDI guest molecules. The chiral arrangement of the right-handed (R)-1NDI molecular units is driven by the chiral cyclohexane-1,2-diamine moiety via hydrogen bonding, and the bulky ethoxy substituents at (R)-1NDI core disfavor strong intermolecular - interaction in the (R)-1NDI assembly, resulting in semi-open -space chiral binding motifs that enable highly selective recognition of (S)-2PDI guests via -surface interaction. In this work, we further investigate possible excitonic delocalization along (R)-1NDI nanofibers on a glass substrate and demonstrate efficient light-harvesting properties towards (S)-2PDI guest molecules. Extensive analysis of their spectral properties, along with significant energy transfer efficiency on individual nanofibers and their anisotropic local environments, is demonstrated using precise wide-field fluorescence microscopy imaging with polarization analysis in combination with atomic force microscopy (AFM).