Abstract: In this paper, the natural antibacterial agent thymol was used as a mildew inhibitor for bamboo, which was immobilized in bamboo by laccase. The mildew resistance of the treated bamboo against Aspergillus niger, Trichoderma virens and Penicillium citrinum was tested, respectively. The laccase-catalyzed fixation effect was evaluated by volatility of thymol, the mildew resistance after leaching treatment and the changes of water contact angle. Scanning electron microscopy (SEM) was used to study the micro-morphology of the treated bamboo, and further Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) were employed to explore the chemical bond changes during the laccase-catalyzed process. The results show that, compared with the bamboo treated by thymol alone, the bamboo treated by laccase catalysis exhibited a good inhibitory effect on A. niger, T. virens and P. citrinum before and after the leaching treatment, the mildew-proof efficiency was above 80%, and the mildew-proof performance of the bamboo treated both with thymol and laccase after the leaching treatment was better than that treated with thymol alone. The volatility of thymol was significantly reduced from 17.03% to 6.75%. The water contact angle increase significantly from 35° to 97°, which was contributing to the mildew resistance of treated bamboo. The enzymatic immobilization did not significantly change the micro-morphology of bamboo, but a large number of flaky substances appeared on the surface of bamboo cell wall, which was considered as aggregates of thymol molecules grafted onto the surface of bamboo. The FT-IR and XPS results shown new formed chemical bonds between thymol and bamboo lignin were the key factor for immobilization of thymol. This study realized the green fixation of natural antibacterial agent through the catalysis of laccase, reducing its volatilization and leaching, and the mildew resistance of treated bamboo was prolonged. The results of this study provide a new idea for green mildew prevention of bamboo, which can achieve safe and long-lasting protection of bamboo.