The selection of effective pretreatment method for exploiting lignocellulosic biomass plays an important role for sustainable biofuel production as it accounts the highest share of production cost. The present study aims to compare the efficiency of glycerol thermal pretreatment (GTP) over hydrothermal and alkali pretreatments of paddy (rice) straw as a feedstock. The efficacy of pretreatment was assessed by analyzing the reducing sugar (RS) yield, holocellulose digestibility and component degradation. The study was further elaborated for enzyme kinetics, FTIR spectra, crystallinity, accurate mass and energy balance. GTP showed 71.52% RS yield with 94.36% holocellulose digestibility, which was higher than that of alkali and hydrothermal pretreatments. GTP was more effective in lignin and hemicellulose removal with little damage to cellulose. The enzyme kinetic study further proved the effectiveness of GTP and showed the enhanced enzyme adsorption and hydrolysis on exposing enzyme binding sites. Crystallinity of cellulose revealed the effectiveness of GTP by XRD studies. FTIR study gives emphasis on alteration in lignocellulose structure due to pretreatment. The mass and energy balances further confirmed the reliability of GTP pretreatment. (C) 2020 Elsevier Ltd. All rights reserved.