The hemicellulose content in holocellulose was adjusted by an alkaline treatment. The effects of this treatment on the defibrillation efficiency of holocellulose nanofibrils (HCNFs) were investigated by wet disk-milling (WDM), along with their morphological and physical properties. In addition, the tensile properties of nanopaper sheets fabricated with these HCNFs were investigated. As the hemicellulose content decreased, the average diameter and the filtration time of the HCNFs decreased, whereas specific surface area and crystallinity index increased. An increase in the WDM time reduced the average diameter and crystallinity of the HCNFs and increased their filtration time and specific surface area. The tensile strength and elastic modulus of the nanopaper sheets increased with increased hemicellulose content and WMD time.