Cellulose nanocrystals (CNCs) were isolated for the first time from pine cones (PC) by alkali and bleaching treatments and subsequent sulfuric acid hydrolysis (64%) at 45 °C. The influence of the hydrolytic reaction time (30, 45, and 90 min) on the yield, chemical composition and structure, and thermal stability of CNCs was evaluated. The removal of non-cellulosic constituents during the alkaline and bleaching treatment resulted in high pure cellulosic fibres. The isolation of CNCs from these cellulosic fibres at different reaction times was verified by the nano-dimensions of the individual crystals (< 3 and < 335 nm of average diameter and length, respectively). The highest yield (15%) and the optimum CNCs properties in terms of aspect ratio, thermal stability and crystallinity were obtained for an extraction time of 45 min. PC appeared to be a new promising source of cellulose fibres and CNCs with potential to be applied as reinforcement in composites and for food-packaging.