Abstract: Dissolved organic matter (DOM) plays an important role in carbon cycling, nutrient supply, and pollutant transport in both natural environments and engineered systems. The main techniques for DOM characterization include ultraviolet-visible (UV-Vis) spectroscopy and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy. This review summarizes the fundamental principles of UV-Vis and 3D-EEM and systematically examines the significance and applications of key optical indices, including absorption coefficients, spectral slope ratio (S_R), fluorescence index (FI), and humification index (HIX). On this basis, the complementary mechanisms and advantages of their combined application are discussed. UV-Vis characterizes DOM aromaticity, molecular weight, and the degree of humification through parameters such as absorption coefficients and S_R, whereas 3D-EEM coupled with parallel factor (PARAFAC) analysis enables the identification of DOM fluorescence components (e.g., humic-like substances) and the evaluation of DOM sources and transformation characteristics using FI, HIX, and related indices. The combined use of UV-Vis and 3D-EEM provides complementary information on DOM structural properties, compositional characteristics, and source apportionment, offering an efficient tool for investigating the physicochemical characteristics and transformation mechanisms of DOM. This integrated approach has been widely applied in freshwater, marine, soil, and water treatment systems to trace DOM sources, migration and transformation processes, and complexation with heavy metals, optimize water treatment processes, and predict the formation potential of disinfection by-products. However, limitations such as spectral overlap, fluorescence quenching effects remain, and interference from environmental variables, and further improvements are required in molecular-level structural interpretation and methodological standardization.