Alkaline fermentation for volatile fatty acids (VFAs) production has shown potential as a viable approach to treat sewage sludge. The hydrolysis and acidogenesis of sludge are greatly influenced by mixing. However, the effects of mixing intensity on VFAs production in sludge alkaline fermentation (SAF) remain poorly understood. This study investigated the impacts of mixing intensity (30, 90 and 150 rpm continuous mixing, and 150 rpm intermittent mixing) on VFAs production, dissolved organic matter (DOM) characteristics, phospholipid fatty acid profiles and microbial population distribution in SAF. Results showed that 150 rpm continuous and intermittent mixing enhanced the hydrolysis of sludge, while 150 rpm intermittent mixing resulted in the highest VFAs production (3886 ± 266.1 mg COD/L). Analysis of fluorescent and molecular characteristics of DOM revealed that 150 rpm intermittent mixing facilitated the conversion of released DOM, especially proteins-like substances, into VFAs. The abundance of unsaturated and branched fatty acids of microbes increased under 150 rpm intermittent mixing, which could aid in DOM degradation and VFAs production. Firmicutes and Tissierella were enriched at 150 rpm intermittent mixing, which favored the maximum VFAs yield. Moreover, Firmicutes were found to be the key functional microorganisms influencing the yield of VFAs during SAF. This study provides an understanding about the mixing intensity effects on VFAs production during SAF, which could be helpful to improve the yield of VFAs. Display omitted •150 rpm continuous mixing led to the highest hydrolysis of sludge.•150 rpm intermittent mixing resulted in the maximum VFAs production.•The enhanced degradation of DOM molecules contributed to VFAs production.•Firmicutes were the key functional microorganisms influencing VFAs yield.