Membrane technologies have attracted growing interest in the fields of water purification and energy recovery. Membrane fouling is the bottleneck that limits the wide application of membrane-based technologies. Exploration of the fouling mechanism and the chemical composition, structure, and functions of foulants and their changes in the membrane filtration process is therefore of significant importance. Molecular spectroscopic methods are among the most efficient platform tools for characterizing membrane fouling because of their unique advantages, such as chemical identification at the molecular level, high sensitivity, and spatially temporally resolved feasibility. Here, an overview of spectroscopic and microspectroscopic approaches, including UV-vis, fluorescence, infrared, and Raman spectroscopy, for the online detection, identification, visualization, and chemical characterization of membrane fouling is provided, and their main advantages as well as limitations in membrane-fouling research are analyzed. Furthermore, future possibilities and challenges for the development of molecular spectroscopy for membrane-fouling characterization are envisaged in this critical review.