Serine is a functional amino acid that effectively regulates the physiological functions of an organism. This study investigates the effects of adding exogenous serine to a culture medium to explore a feasible method for the rejuvenation of V. volvacea degenerated strains. The tissue isolation subcultured strains T6, T12, and T19 of V. volvacea were used as test strains, and the commercially cultivated strain V844 (T0) was used as a control. The results revealed that the addition of serine had no significant effect on non-degenerated strains T0 and T6, but could effectively restore the production characteristics of degenerated strains T12 and T19. Serine increased the biological efficiency of T12 and even helped the severely degenerated T19 to regrow its fruiting body. Moreover, exogenous serine up-regulated the expression of some antioxidant enzyme genes, improved antioxidase activity, reduced the accumulation of reactive oxygen species (ROS), lowered malondialdehyde (MDA) content, and restored mitochondrial membrane potential (MMP) and mitochondrial morphology. Meanwhile, serine treatment increased lignocellulase and mycelial energy levels. These findings form a theoretical basis and technical support for the rejuvenation of V. volvacea degenerated strains and other edible fungi.