The objective of this study was to examine the impacts of the combing of Agrocybe aegerita polysaccharides (AAPS) with Bifidobacterium lactis Bb-12 (Bb-12) on antioxidant activity, anti-aging properties, and modulation of gut microbiota. The results demonstrated that the AAPS and Bb-12 complex significantly increased the average lifespan of male and female Drosophila melanogaster under natural aging conditions (p < 0.05), with an improvement of 8.42% and 9.79%, respectively. Additionally, the complex enhanced their climbing ability and increased antioxidant enzyme activity, protecting them from oxidative damage induced by H2O2. In D-galactose induced aging mice, the addition of AAPS and Bb-12 resulted in significantly increase in antioxidant enzyme activity, regulation of aging-related biomarker levels, changed gut microbiota diversity, restoration of microbial structure, and increased abundance of beneficial bacteria, particularly lactobacilli, in the intestines. These findings suggested that the complex of AAPS and Bb-12 had the potential to serve as a dietary supplement against organism aging and oxidative stress.