Summary: A plethora of biological processes hinge on the intricate dance of biomolecular interactions. Typically, scientists use the traditional fluorescence co-localization method to investigate how biomolecules interact in close proximity. However, there's a catch – this method has a resolution limit of around 200nm. In this tiny space, millions of molecules can be crammed together, making it tricky to confirm if the observed signals truly indicate interactions between biomolecules. To address this challenge, we introduce a novel spectrally-resolved fluorescence microscopy technique centered on energy transfer (ET) to validate these biomolecular interactions. The beauty of ET lies in its sensitivity to distance – it significantly changes and becomes negligible after a few molecular dimensions. This allows us to measure sensitized emission, proportionate to the extent of ET, providing a reliable method to explore local interactions among biomolecules labeled with donors and acceptors. Our technique has already shown promising results in practical applications. We successfully demonstrated its effectiveness in studying phase-separated α-synuclein droplets, the local surface polarity of α-Syn fibril and investigating supramolecular block copolymers featuring alternate donor- and acceptor-labeled segments.