Recently, cellulose nanocrystals (CNs) with various morphologies (rod-like or spherical) isolated from the most abundant cellulosic materials have gained increasing attention in some fields such as antimicrobial packagings [1,2], template for metallic nanoparticles [3], aerogels for wastewater treatment [4], drug delivery [5], and protein immobilization [6] due to their excellent properties, including easily modified functional groups, high specific area, aspect ratios, and outstanding stiffness. As the nanofillers, the requirement of CNs surface group varies with the different applications. The traditional methods like acid hydrolysis [4,7], enzyme-assisted hydrolysis [8] and mechanical treatments [9] can extract the CNs with hydroxyl groups, which are usually modified into other functional groups for further applications. Recently, CNs with functional groups (carboxyl or aldehyde groups) have been developed by 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) [10,11], ammonium persulfate (APS) [12], two-step approach combined acid hydrolysis and NaIO 4 oxidations [13][14][15], and one-step NaIO 4 oxidation [16]. The TEMPO and APS oxidations consume for a long time and the oxidizing agents are toxic [10,12] Abstract. A novel double response surface model is used first time to optimize a regioselective process to prepare spherical dialdehyde cellulose nanocrystals (SDACN) and rod-like dialdehyde cellulose nanocrystals (RDACN) via one-step sodium periodate (NaIO 4 ) oxidation. The influence of four preparation factors (solid-liquid ratio, NaIO 4 concentration, reaction time and temperature) on the yields and aldehyde contents of the final products were evaluated. For comparison, rod-like cellulose nanocrystals (CN-M and CN-S) were prepared by hydrochloric/formic acid hydrolysis and sulfuric acid hydrolysis, respectively. The RDACN shows high crystallinity of 82%, while SDACN presents low crystallinity due to the high degree of oxidation. Thus, SDACN has poorer thermal stability than RDACN and CN-M, but higher than CN-S. Compared to CN-M, SDACN with higher aldehyde contents as templates is beneficial to deposit more Ag nanoparticles with diameters of 30±4 nm and the resultant nanohybrids exhibit good antibacterial activities against both Gram-negative E. coli and Gram-positive S. aureus.