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Abstract. Kapok is a source of natural fiber in addition to cotton, which availability is abundant and contain high cellulose but not been utilized optimally. This study aimed to determine the optimal conditions of sulfuric acid concentration and hydrolysis time in the production process of nanocrystalline cellulose (NCC) from kapok fiber. The research was conducted in several stages, namely the delignification with alkali hydrothermal, bleaching with alkaline hydrogen peroxide agents, isolation with sulfuric acid solutions, and NCC characterization. The optimization used Response Surface Methodology (RSM) with Central Composite Design (CCD), which consisted of two factors, namely H2SO4 concentration and hydrolysis time. The optimized parameter was the crystallinity degree of the NCC. NCC characterization included functional group analysis with Fourier Transform Infrared Spectroscopy (FTIR), surface profiles and dimensions by Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). The results showed that the optimum condition of NCC isolation from kapok fiber was obtained at 54.46% (b/b) H2SO4 with hydrolysis time of 48.96 minutes, resulting in a maximum degree of crystallinity of 71.8%. The hydrolysis process with H2SO4 caused a change in the NCC functional group. NCC had a diameter of about 11.2 nm and high thermal stability that potential for various composite materials