This study investigated the impact of pretreatment temperature on the dissolution of organic matter in sludge pretreated with alkaline rhamnolipid and its subsequent effect on the microbial community structure during anaerobic fermentation. The findings revealed that when the pretreatment temperature reached 80 °C, the maximum concentration of SCOD is 5008.89 mg/L, which is 4 times higher than that achieved at room temperature. Three-dimensional fluorescence parallel factor analysis indicated that increasing the pretreatment temperature significantly enhanced the degree of humification of sludge DOM and reduced the ratio of protein-like and fulvic acid in the sludge. The 16S rRNA gene-based analyses revealed that the relative abundance of Bacteroidetes was highest at 80 ℃ (11.14 %), and continued to increase as fermentation time progressed. This study highlights the critical role of temperature in the three-factor pretreatment of sludge, demonstrating that higher pretreatment temperatures not only accelerate the acid production rate during anaerobic fermentation but also enhance overall acid production.