Freshly harvested Tremella fuciformis contains high water content with an unprotected outer surface and exhibits high respiration rates, which renders it prone to moisture and nutrient loss, leading to decay during storage. Our research utilized ε-poly-L-lysine (ε-PL) and chitosan as a composite coating preservative on fresh T. fuciformis. The findings revealed that the ε-PL + chitosan composite coating preservative effectively delayed the development of diseases and reduced weight loss during storage compared to the control group. Furthermore, this treatment significantly decreased the respiration rate of T. fuciformis and the activity of respiratory metabolism-related enzymes, such as alternative oxidase (AOX), cytochrome c oxidase (CCO), succinic dehydrogenase (SDH), 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase (6-PGDH and G-6-PDH). Additionally, the composite coating preservative also delayed the depletion of ATP and ADP and maintained higher levels of the energy charge while preserving low levels of AMP. It also sustained heightened activities of Mg2+-ATPase, Ca2+-ATPase, and H+-ATPase enzymes. These results demonstrate that utilizing the ε-PL + chitosan composite coating preservative can serve as a sufficiently safe and efficient method for prolonging the shelf life of post-harvest fresh T. fuciformis.