Fluorescence spectrometry (FLS) is a fundamental toolbox that provides in-depth information about the electronic transitions and interactions in a molecular system. With this technique, one can assay the state of a physical, chemical, or biological system by studying their photophysical properties. The electronic transitions or interactions in a particular molecular system are monitored by analyzing the changes in the fluorescent optical properties and their life-times. In this chapter, we have included the fundamental principles of fluorescence spectrometry along with the experimental and working aspects. The concepts of steady-state and time-resolved fluorescence spectroscopy and the modern development in this regime have been discussed in detail. All the aspects of FLS for characterizing the soft-nano composites have been comprised and explained. The future perspectives and modern applications of FLS in physical, chemical and medical sciences along with the bio-imaging have been covered within the scope of this chapter.