Cellulosic fibers from the bast of Pueraria lobata (P. lobata) vine were separated using a “green” and efficient method that combined steam explosion (SE) and a laccase mediator system (LMS). The chemical components, structure, and thermal alterations in the fibers were evaluated. The SE performed at 180 °C for 10 min did not change the chemical composition of P. lobata; however, SE did alter the fiber structure and rendered its surface more accessible to the laccase enzyme. Treated and untreated samples were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD), thermogravimetric analysis (TGA), and chemical methods. The cellulose content of the processed fibers was approximately 68.2%, and the lignin content was 11.8%, which was much lower than the 22.98% lignin content of the raw material. The cellulose fibers exhibited higher cellulose crystallinity and thermal stability compared with the untreated samples. This combined treatment approach may be useful for the isolation of cellulose fibers for composites, textiles, and other industrial applications.