Anaerobic digestion is an important approach for reducing the volume and realizing resource recovery from urban sludge. However, traditional anaerobic digestion is limited by the diffusion of hydrogen/formic acid, easily leading to metabolic blockage and ultimately acidification, and causing system collapse. Direct interspecies electron transfer (DIET) has been proven to effectively mitigate these problems and improve the efficiency of anaerobic digestion. Research shows that adding a small amount of glycerol can enrich electroactive microorganisms, promoting DIET. However, exogenous glycerol addition increases operating costs. Therefore, this experiment combined alkali pretreatment and yeast fermentation to produce glycerol in situ from urban sludge, promoting DIET and providing technical support for efficient methane production from urban sludge. The research results showed that: (1) The optimal parameter for alkaline pretreatment of urban sludge was 10 hours. This increased the soluble sugar content in the urban sludge by 43.4% compared to untreated sludge. (2) The optimal inoculation yeast amount was 10%, and the optimal fermentation period was 9 hours. Glycerol concentration accounts for 2.43% of the total chemical oxygen demand (COD) of urban sludge. (3) Anaerobic digestion performance of urban sludge was evaluated based on indicators such as methane yield, volatile solid (VS) removal rate, and organic matter conversion efficiency. Compared with the control group, the yeast fermentation group and the alkaline pretreatment combined with yeast fermentation group showed increases in methane production of 11.8% and 15.4%, respectively. The removal rate of volatile solids was similar in all group (approximately 45%), while organic matter conversion efficiency increased by 5.80% and 9.30%, respectively. (4) By testing the electron transfer coefficient (ETC) of the control group, yeast fermentation group, and alkaline pretreatment combined with yeast fermentation group, the intrinsic promotion mechanism of the system was revealed. Compared with the control group, the ETC of discharge in the yeast fermentation group and the alkaline pretreatment combined yeast fermentation group increased by 11.1% and 16.8%, and the ETC of charging increased by 11.1% and 17.3%, respectively. (5) Changes in microbial community caused by alkaline pretreatment combined with yeast fermentation treatment of urban sludge was revealed through microbial community analysis. The analysis showed that Methanothrix soehnenii GP6 and Fastidiosipila sanguinis was enriched in the alkaline pretreatment combined with yeast fermentation group, which may be involved in DIET, compared to the control group.