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[Display omitted] • Autohydrolysis enhanced the delignification of p -TsOH post-treatment of poplar. • Unexpectedly, the glucose yields of combined pretreated poplar decreased. • The intensified surface lignin coverage hindered the enzymatic hydrolysis. • Tween 80 enabled the excellent enzymatic hydrolysis of combined pretreated poplars. In this study, a combined pretreatment involving autohydrolysis and p -toluenesulfonic acid (p -TsOH) was performed on poplar to coproduce xylooligosaccharides (XOSs) and monosaccharides. The autohydrolysis (180 °C, 30 min) yielded 53.2 % XOS and enhanced the delignification efficiency in the subsequent p -TsOH treatment. Furthermore, considerably high glucan contents (64.1 %∼83.1 %) were achieved in the combined pretreated substrates. However, their enzymatic digestibilities were found to be extremely poor (9.6 %∼14.2 %), which were even lower than the single p -TsOH pretreated substrates (10.2 %∼35.8 %). The underlying reasons were revealed by systematically investigating the effects of the single and combined pretreatment strategies on substrate properties. Moreover, the Tween 80 addition successfully reversed the adverse effects of combined pretreatment on the enzymatic hydrolysis, achieving a high glucose yield of 99.3 % at an enzyme loading of 10 filter paper units/g (FPU/g) glucan. These results deepen the understanding of the synergy of combined pretreatment on biomass fractionation and enzymatic saccharification. [ABSTRACT FROM AUTHOR]