: This review attempts to cover the principles and environmental applications of excitation-emission spectrofluorimetry (EES). The article is divided into two parts and each part is divided into several sections. The first part includes the following: Introduction, advantages and drawbacks of EES, comparative EES versus other techniques, factors influencing EES signals, representation of EES spectra, relationships and changes in intensity of fluorescence, multivariate calibration combined with EES, quenching of fluorescence, and conclusions. Theoretical and practical considerations are included, and the possibilities and limitations are evaluated. The second part is devoted to the environmental applications of EES: Characterization of chromophoric dissolved organic matter (CDOM) in drinking waters, rivers, fog water, lakes, oceans, leachates, wastewaters, sludge, bioreactor membrane foulants and soils; characterization of extracellular polymeric substances in sluges; study of interactions between CDOM and organic pollutants in soils; and quantification of organic pollutants in waters and soils. This part includes the pretreatment of samples, and the environmental applications of the technique are discussed, including its application to biorremediation of wastewaters. More than 500 references (focusing mainly on the last 10 years) from all kinds of journals (environment, analytical chemistry, biotechnology) have been critically reviewed and included in this article.