Wastewater treatment plant effluent is considered an important hotspot of dissolved organic matter. The behavior and transformation of dissolved effluent organic matter (dEfOM) regulate particle sedimentation, pollutant fate, microbial attachment, and biofilm formation. However, studies have so far focused on the transformation of marine and riverine organic matter, and the current knowledge of dEfOM behavior is still limited. Fluctuations in water conditions, especially temperature, may directly alter the size, assembly speed, and structure of microgels, thereby potentially disturbing fate and the transportation of organic matter. In this study, we firstly investigated the effects of temperature on the behavior and capacity of dEfOM assembly into microgels and the possible mechanism. The microgel size and granularity of dEfOM were monitored by flow cytometry. Our results suggest that, with regard to microgels, a higher temperature leads to a higher assembly capacity but also a decrease in the size distribution. By contrast, assembly at 4 °C reduces the relative assembly capacity but increases the microgel size and granularity. The size distribution of the formed microgels at the various temperatures was ordered as follows: 4 °C > 20 °C > 35 °C. The size reduction in dEfOM assembly may be closely tied to the enhancement of hydrophobic interactions. The reduction in microgel granularity in warm conditions (35 °C) in terms of the effluent water may be caused by thermally induced condensation. Overall, the findings demonstrate the effects of temperature on dEfOM assembly and can facilitate further relevant studies on aquatic organic particle formation during current global warming scenarios.