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 (NaIO4) oxidation. The influence of four preparation factors (solid-liquid ratio, NaIO4concentration, 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.