This chapter introduces the fundamentals, principles, and data analysis of Spectral Self-Interference Fluorescence Microscopy (SSFM). It discusses some of the applications in biosensing and bimolecular studies on the SSFM platform. Conventional tools are available to study DNA–protein binding whose critical size dimensions are on sub-nanometer scales. From the spectral oscillations emitted by an ensemble of fluorophores located above a reflecting interface, the average height of the fluorophores relative to the surface can be determined with sub-nanometer resolution across a broad range from a few nanometers to more than 100 nm. The direct contact of molecules to the interface as well as other local steric conditions will affect the conformations of the biomolecules, which potentially influence their functions in binding. The requirement to adsorb the molecules on a surface impacts the conformation of the adsorbed biomolecules, and scanning of a surface by atomic force microscopy is a slow process.