**Abstract**

This chapter summarizes the fundamentals of biosensing techniques based on fluorescence spectroscopy and the protagonism of state-of-the-art luminescent biosensors in a wide range of scientific areas, from environmental monitoring to diagnostics and decease treatment, focusing on the paramount contribution of biosensors based on the Förster Resonance Energy Transfer (FRET) transducing mechanism. State-of-the-art FRET biosensors are specially characterized by outstanding sensitivity toward a number of environmental pollutants and dissolved oxygen in aquatic ecosystems, capable of detecting concentrations in the nano and picomolar scales. These biosensors have also been showing impressive performance over other methods in the study of real-time biological processes *in vivo* relevant to help understanding decease progression like cancer.

**Keywords:** fluorescent biosensors, ROS detection, sensitivity, FRET biosensors, new materials
