The pretreated biomass was further utilized to obtain levoglucosan by fast pyrolysis. The pretreated sugarcane bagasse exhibited significantly higher levoglucosan yield (47.70%) than that of un-pretreated sample (11.25%). The promotion could likely be attributed to the effective removal of alkali and alkaline earth metals by glycerol pretreatment. This research developed an economically viable manufacturing paradigm to utilize glycerol comprehensively and enhance the formation of levoglucosan effectively from lignocellulose.