This study aimed to evaluate the effect of ammonium persulfate’s (APS) oxidation time on the characteristics of the cellulose nanocrystals (CNCs) of balsa and kapok fibers after delignification pretreatment with sodium chlorite/acetic acid. This two-step method is important for increasing the zeta potential value and achieving higher thermal stability. The fibers were partially delignified using acidified sodium chlorite for four cycles, followed by APS oxidation at 60 °C for 8, 12, and 16 h. The isolated CNCs with a rod-like structure showed an average diameter in the range of 5.5–12.6 nm and an aspect ratio of 14.7–28.2. Increasing the reaction time resulted in a gradual reduction in the CNC dimensions. The higher surface charge of the balsa and kapok CNCs was observed at a longer oxidation time. The CNCs prepared from kapok had the highest colloid stability after oxidation for 16 h (−62.27 mV). The CNCs with higher crystallinity had longer oxidation times. Thermogravimetric analysis revealed that the CNCs with a higher thermal stability had longer oxidation times. All of the parameters were influenced by the oxidation time. This study indicates that APS oxidation for 8–16 h can produce CNCs from delignified balsa and kapok with satisfactory zeta potential values and thermal stabilities.