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Graphical abstract Highlights • Enzyme and cold alkali assisted mechanical production of CNF was studied. • Thermal stability of CNF will be affected by cellulose crystalline structure. • Presence of hemicellulose in CNF improves their thermal stability. • Presents new avenues for research on CNF-based electronic devices. Abstract Lignocellulosic biomass is the most abundant renewable resource on the earth. With the development of related fields, the high value utilization of lignocellulosic biomass has gradually become a new avenue for research. In this study, unbleached bagasse pulp was pretreated with xylanase and cold alkali to partially remove hemicellulose and convert to some cellulose I into cellulose II. Cellulose nanofibrils (CNF) were then obtained through ultra-micro grinding and high-pressure homogenization. The prepared CNF were characterized by TEM, Zeta potential, ATR-FTIR and XRD, and a thermogravimetric analyzer was used to analyze the thermal stability of CNF. The results show that xylanase pretreatment can improve the dispersion of fibers during mechanical treatment and can enhance the crystallinity of CNF. With an increase in alkali concentrations, the proportion of cellulose II structures increased, while cellulose crystallinity levels decreased due to the folding of cellulose chains. Under the common influence of crystallinity and crystal structures, the thermal stability of the CNF prepared after cold alkali pretreatment underwent an increasing trend. This shows that the influence of crystal structures on the thermal stability of CNF gradually plays a dominant role as alkali concentrations increase. [ABSTRACT FROM AUTHOR]