Sewage sludge (SS) alkaline fermentation (especially at pH 10) can efficiently enhance volatile fatty acids (VFAs) production. VFAs are considered an excellent carbon source for the biological nutrient removal (BNR) process. Dissolved organic matter (DOM) in fermentation liquid is the direct substrate used for producing VFAs and can greatly influence the effluent quality of BNR process. However, knowledge of DOM characteristics in sludge alkaline fermentation is limited. This study focused on the functional groups, fluorescent components and molecular features of DOM as well as molecular weight of proteins in SS alkaline fermentation (at pH uncontrolled, 7, 8, 9 and 10). Results showed a significantly improved generation of tryptophan-like and tyrosine-like substances as well as molecular weight <1 kDa proteins (381.5 ± 38.4 mg/L) was observed at pH 10 (p < 0.05). Further analysis of DOM molecular characteristics indicated that pH 10 resulted in the highest molecular diversity and the generation or degradation of easily biodegradable lipid-like and proteins/amino sugars-like formulas. The improved solubilization of DOM contributed to VFAs production. Meanwhile, increasing pH to 10 also promoted the release of hard-biodegradable organic matter, e.g., humic-like and lignin-like substances. Additionally, a high diversity of resistant N-containing organic molecules was generated at pH 10. Fermentation of SS at pH 10, is favored to enhance VFAs production and, can also result in a higher content of refractory DOM. This study helps to achieve a comprehensive understanding of SS alkaline fermentation and provides fundamental information for further treatment.