The 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)/laccase/O-2 system was used to prepare cellulose nanofibers from wood cellulose without requiring any chlorine-containing oxidant. Lactase was degraded by oxidized TEMPO (TEMPO+) formed by laccase-mediated oxidation with O-2, which competed with the oxidation of wood cellulose. Thus, large amounts of laccase and TEMPO and a long reaction time were needed to introduce similar to 0.6 mmol g(-1) of C6-carboxylate groups onto wood cellulose. The TEMPO/laccase/O-2 system underwent one-way reaction from TEMPO to reduced TEMPO through TEMPO+. When the oxidation was applied again to the oxidized wood cellulose following isolation and purification, the C6-carboxylate groups increased to similar to 1.1 mmol g(-1), which was sufficient to convert the sample to cellulose nanofibers by sonication in water. However, the higher the carboxylate content of the oxidized celluloses, the lower their degree of polymerization.