Ammonium persulfate (APS) oxidation was employed to isolate carboxyl cellulose nanocrystals (CNCs) from hybrid poplar residue. Structure changes resulting from APS oxidation were investigated by Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). The further oxidation of the ensuring CNCs with sodium periodate manifested selective oxidation of hydroxyl groups at the C6 position of cellulose into carboxyl groups during APS oxidation. The introduction of active carboxyl groups resulted in lower thermal stability. Transmission electron microscopy (TEM) and width distribution showed that the produced CNCs ranged from 10 nm to 24 nm. Carboxyl CNCs with a yield of 63.2% were isolated via APS oxidation, and they were suitable for large-scale CNCs production.