Cellulose nanocrystals are promising materials for application in biomedicine, pharmaceutical and food industry. There are various methods for CNC synthesis, but acid hydrolysis is the most common one. The optimization of acid hydrolysis process is still in progress. In the present study, different CNC suspensions were prepared to find out the more efficient acid ratio for CNC production. Whatman #1 filter paper was hydrolyzed via 64 wt% sulfuric acid at acid-to-paper ratio of 10:1 mL/g, 20:1 mL/g, 30:1 mL/g, and 40:1 mL/g. The synthesized CNCs were characterized by DLS, ELS and AFM. The suspensions with acid/cellulose ratio of 20:1 mL/g & 30:1 mL/g resulted in CNCs with more negative surface charge and uniform size. They also represented chiral nematic phase and regular self-organization after layer by layer deposition. It was found that the higher acid-to-paper ratio does not necessarily produce the high quality CNCs. Since the suspension that was prepared at acid/cellulose ratio of 40:1 mL/g had lower negative charge. The size and surface charge of CNCs are highly dependent to acid hydrolysis parameters and ultimately affect their behavior in nano-composites. In spite of many published papers around CNC synthesis by acid hydrolysis, there are still some details that must be addressed and investigated more to prepare the most efficient and applicable CNCs. Manuscript profile