Fly ash derived from the incineration of garbage is known to contain hazardous materials that can affect the growth of plants and animals and pose a threat to human health. In this study, we explored how treatment of fly ash leachate with microalgae could alter the properties of dissolved organic matter (DOM). Fly ash leachate samples obtained from a landfill site in Wenzhou were treated with the microalgae Chlorella vulgaris or Scenedesmus obliquus without and with the addition of ammonium ferric citrate (C6H8FeNO7) for 24 days, and changes in DOM levels and types were measured using excitation emission matrix fluorescence technology. The following results were obtained: Analysis of three-dimensional fluorescence spectral indices indicated that the algal treatment process consistently generated new autogenous DOM, with most of the organic matter being newly formed. Additional nutrients had a minor effect on the production and composition of DOM in the system. Using a parallel factor model to analyze the three-dimensional fluorescence spectral matrices of water samples from various systems revealed common components in each group, including arginine, tryptophan-like proteins and fulvic acid-like substances. This study aimed to explore the changes in DOM properties during microalgae treatment of fly ash leachate from the perspective of three-dimensional fluorescence.