Seed aging is always taken as a crucial factor for vigor loss due to delayed seed germination and seedling growth, which limits hay production. Many studies have found that telomeres are closely related to abiotic stress and seed vigor. However, the molecular mechanism of telomeres’ response to abiotic stress, seed vigor, and the maintenance mechanism of plant telomere homeostasis still remain unclear. Alfalfa (Medicago sativa) enjoys the title of “King of Forage”, and is an important protein forage for the dairy industry as planted in the world. This comprehensive investigation was performed to explore the molecular characterization, phylogenetic relationship, and gene expression analysis ofMsTERTunder abiotic stress and during seed aging in alfalfa. In this study,MsTERTwas identified from the ‘Zhongmu 1’ alfalfa genome and encoded a coding sequence (CDS) of 3615 bp in length, consisting of telomerase- RNA-Binding Domain (RBD) and Reverse Transcriptase (RT) domains, 1024 amino acids, an isoelectric point of 9.58, and a relative molecular mass of 138.94 kD. Subcellular localization showed thatMsTERTwas mainly localized in the nucleus and mitochondria. The results of the expression profile showed thatMsTERTwas observed to respond to various stress conditions such as salt (100 mmol/L NaCl) and drought (20% PEG 6000). Furthermore, exogenous hormones IAA, ABA, and GA3showed the potential to affectMsTERTexpression. Additionally,MsTERTalso responded to seed aging. Our results revealed a marginal but significant association between relative telomere length,MsTERTexpression, and seed germination percentage, suggesting that the length of telomeres was shortened, and expression ofMsTERTdecreased with alfalfa seed aged. These results provide some evidence for the hypothesis of relative telomere length and/or TERT expression serving as biomarkers of seed aging. Although this finding is helpful to offer a new way to elucidate the molecular mechanism of vigor loss in alfalfa seed, further investigation is required to elucidate the molecular mechanism by which theMsTERT generegulates seed vigor.