•The extraction of cellulose nanocrystals (CNC) has been carried out from hemp fibres.•Carded and not carded fibres were chemically and enzymatically pre-treated.•Carding allowed removal of most impurities from the fibres.•CNC were extracted by acid hydrolysis from pre-treated carded hemp.•The enzymatic treatment resulted in major changes in X-ray diffraction of carded hemp. The extraction of cellulose nanocrystals (CNC) from Carmagnola hemp fibres has been carried out. Before CNC extraction, the effectiveness of two pre-treatment methods, an alkaline chemical and a pectinase enzymatic treatment, applied on the pristine and carded hemp fibres, were compared. Carding allowed removal of most impurities from the fibres, while it had only a modest effect on their structure. After chemical treatment, hemicellulose was removed, more efficiently in carded hemp, and X-ray diffraction suggests an increase in the size of cellulose crystallites. Carded hemp fibres, after interaction with pectinase, show the total decomposition of pectin and hemicellulose. On the basis of these results, carded hemp was selected as start material for CNC extraction and the acid hydrolysis for CNC synthesis was applied on carded hemp after both chemical and enzymatic procedures. The yield of the different hydrolysis methods remains approximately at the same level (around 19%): on the other side, a carding procedure, combined with an alkaline treatment to hemp fibres, represents an optimized process for CNC extraction by acid hydrolysis.