In this paper, using Styphnolobium japonicum, Cryptomeria fortunei and Pinus yunnanensis as raw materials, cellulose (CE) was first extracted by chemical method (CM1) and biological enzymatic method 2 (BM2), and then further prepared into nanocellulose fiber (CNF) by biological enzymatic method 1(CPM (CPM stands for cellulase and pectinase)). The CE and CNF were characterized by FTIR, XRD, SEM, DSC and TG. The results showed that the CNF prepared by CM1-CPM was cellulose type II, CNF prepared by BM2-CPM was cellulose type I. The best crystallinity of the CNF prepared by CM1-CPM reached 74.1% ( C. fortunei CNF), the best yield was 24.0% ( S. japonicum CNF) and the minimum average diameter was 62.6 nm ( C. fortunei CNF). However, the best crystallinity of CNF prepared by BM2-CPM reached 80.1% ( C. fortunei CNF), the best yield was 27.4% ( C. fortunei CNF), and the minimum average diameter was 31.6 nm ( C. fortunei CNF). And also, the thermal degradation analysis of the CNF prepared from the three raw materials revealed that the CNF prepared by BM2-CPM had better thermal degradability. Finally, the thermal stability analysis of the C. fortunei CNF was carried out, and it was found that the residual mass of CNF prepared by BM2-CPM was 48%, while the residual mass of C. fortunei CNF prepared by BM1-CPM was 22%, which showed the C. fortunei CNF prepared by BM2-CPM had better thermal stability. Therefore, the properties of nanocellulose prepared by BM2-CPM are optimal.