Sarcopenia is an age-related progressive disease characterized by the loss of skeletal muscle mass and function, which seriously threatens the quality of life and survival of elderly individuals. Shiquandabu Decoction is a classical traditional Chinese herbal formula known for its effects in benefiting Qi and nourishing blood, and modern studies have demonstrated its potential therapeutic effects on sarcopenia. To investigate the protective effects and underlying mechanisms of Shiquandabu Decoction against sarcopenia, this study prepared its water decoction, administered it to rats by gavage, collected the drug-containing serum, and identified the components absorbed into the blood using UHPLC-Q-TOF-MS. The animal experiments in this study were conducted in compliance with the regulations of the Scientific Research Ethics Committee of Shanxi University of Chinese Medicine and approved by the Animal Experimentation Ethics Review (approval No.: AWE202403229). The drug-containing serum was used to treat a D-galactose (D-gal)-induced senescent C2C12 myotube model. CCK-8 was used to detect cell activity, DCFH-DA probe was used to detect reactive oxygen species (ROS) levels, JC-1 staining was used to detect mitochondrial membrane potential, flow cytometry was used to analyze the cell cycle, and RT-qPCR was used to detect the expression of myogenic differentiation-related factors (MyoD, MyoG, and Mstn) and senescence gene (p53). LC-MS proposed targeted metabolomics to screen for differential metabolites and analyze related pathways. The results showed that 15% drug-containing serum was able to significantly increase the cell survival rate, reduce the ROS levels, restore the mitochondrial membrane potential, reduce the G0/G1 phase cellular block, and promote the cell cycle process, and it was able to up-regulate the expression of MyoD and MyoG, and down-regulate the expression of Mstn and p53. Metabolomics studies of the proposed target cells showed that intervention with drug-containing serum resulted in the callback of 119 differential metabolites (42 up-regulated and 77 down-regulated), mainly related to amino acids, carbohydrates, lipids, nucleotides and energy metabolic pathways. These results suggest that the drug-containing serum of Shiquandabu Decoction exerts a protective effect against D-gal-induced myocyte attenuation through multi-target and multi-pathway synergistic effects, improving mitochondrial function, regulating the cell cycle and promoting myo-differentiation.