[Objective] Meadow soils on the Qinghai-Xizang Plateau are highly sensitive to changes in the frequency of seasonal freeze-thaw cycles caused by climate change，and it is therefore urgent to understand how microbial activity in these soils responds to different numbers of freeze-thaw cycles. [Methods] Typical alpine meadow soil from a region of the Qinghai-Xizang Plateau with frequent seasonal freeze-thaw activity was selected as the study subject. Simulations with 1，3，5，7，and 9 freeze-thaw（FT）cycles（termed C1，C3，C5，C7，and C9）and a non-freeze-thaw control（N9）were conducted to compare the response characteristics of typical soil parameters：dissolved organic carbon（DOC），microbial biomass carbon and nitrogen，and gaseous carbon and nitrogen. [Results] 1）Compared with the non-FT treatment（N9），soil microbial biomass carbon（SMBC） decreased significantly by 23.64% after C9，while DOC increased significantly by 7.37%，and the cumulative emissions of CO₂ and N₂O increased by 71.09% and 321.40%，respectively. 2）With increasing FT cycles，DOC content and activities such as β -glucosidase and leucine aminopeptidase showed a nonlinear trend：they first declined and reached minimum levels at C5. The CO₂ emissions rates peaked after the first two freeze-thaw cycles （1.83 times that of the non-FT soil），while N₂O emission rates sharply increased after the fifth cycle，reaching 5.83 to 24.82 times that of the non-FT soil. 3） Soil microbial biomass nitrogen （SMBN） did not vary significantly，but the concentrations of NO₃^--N and the activity of N-acetylglucosaminidase（NAG）increased steadily，while NH₄⁺-N decreased steadily，as the number of FT cycles increased. [Conclusion] The effects of multiple freeze-thaw cycles on soil microorganisms are not merely repetitions of a single freeze-thaw event. After five cycles，changes in pore structure and microbial mortality caused by soil water freezing and swelling have largely stabilized，and the surviving microbial communities gradually adapt to the new temperature conditions， restoring their activity during subsequent cycles. These findings could provide new theoretical insights and data for understanding the overwintering mechanisms of microorganisms in alpine meadow soils affected by seasonal freeze-thaw cycles.