A novel pretreatment method combining thermophilic bacteria (TB) with alkyl polyglucose (APG) was employed to pretreat waste sludge for enhancing the sludge hydrolysis. TB combined with APG pretreatment was effective in the releasing of soluble chemical oxygen demand (SCOD), protein and carbohydrate in extracellular polymeric substances (EPS) when the dosage of APG was below 0.1 g/g TSS. The enhancement of SCOD, carbohydrates and protein in dissolved organic matter (DOM) was promoted by the synthetic effect of APG and TB, which provides more carbon and energy source to the subsequent biochemical processes in sludge digestion. Excitation-emission matrix (EEM) fluorescence spectroscopy revealed that the combined pretreatment was beneficial for the decrease of non-biodegradable materials and the increase of biodegradable materials in DOM, resulting in the enhancement of the biodegradation of waste sludge. The combined use of TB and 0.4 g/g TSS APG achieved the maximal activities of protease (1.8) and α-glucosidase (1.9), and the activities of protease and α-glucosidase were positively correlated to the dosage of APG. The combined pretreatment was advantageous for the sludge reduction and sludge stabilization.