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Nanocellulose crystals (NCCs) were successfully prepared via acid hydrolysis from an abundant agricultural waste (tea stalk) in China. The effective factors for NCC yield were modeled by the response surface methodology (RSM). The RSM determined the reaction conditions (H 2 SO 4 concentration, hydrolysis temperature, and reaction time) that optimized the yield of tea stalk NCCs (TNCCs). Under the optimized operating conditions, the fundamental properties of TNCCs were characterized via transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), laser diffraction particle-size analyzer, and scanning probe microscopy (SPM). Wood NCCs (WNCCs) and microcrystalline NCCs (MNCCs) were simultaneously prepared from common wood and microcrystalline cellulose under the same conditions. The results show that TNCCs not only shows similar physical and chemical properties with WNCCs and MNCCs, but also has better stability. Therefore, this study offers novel routes for high-valued utilization of tea stalk and provides some theoretical guidance for utilizing cellulose obtained from tea stalk. Unlabelled Image • Successful preparation of Nanocellulose crystals (NCCs) from Tea stalk of agricultural waste; • Conditions for determining the optimal yield of hydrolysis by response surface methodology (RSM); • Nanocellulose crystals were prepared from wood and microcrystal cellulose under the same conditions. [ABSTRACT FROM AUTHOR]