The precise determination of rest-grazing periods is of great significance for the sustainable utilization and scientific management of cold-season pastures in alpine meadow. Soil extracellular enzyme activities and microbial metabolic limitations serve as critical parameters for assessing soil quality, yet their response patterns to different spring rest-grazing periods in alpine meadows of the eastern Qilian Mountains remain unclear. This study used the traditional local rest-grazing period as the control (CK), spring rest-grazing treatments were initiated at rest-grazing soil surface began to thaw (ST1), soil thawing depth was >10 cm (ST2), plant upper ground re-greening (coverage reached 30-40%) (RG1), re-green plant coverage reached 80% (RG2). Soil physicochemical properties and the soil carbon (C), nitrogen (N), and phosphorus (P) acquisition enzymes activities were measured on 1 June (the local traditional rest-grazing time) and 28 July (the vigorous grass growing season) in 2022, we evaluated microbial metabolic limitations using soil enzymatic stoichiometry. The results showed that rest-grazing during the soil thawing periods (ST1 and ST2) significantly inhibited soil C-acquiring enzyme activities (β-1,4-glucosidase and β-D-cellobiosidase), N-acquiring enzyme activities (β-1,4-N-acetylglucosaminidase and L-leucine aminopeptidase), and P-acquiring enzyme activity (acid phosphatase), as well as significantly decreased enzyme C:P and N:P ratios in both June and July. Moreover, vector analysis showed that rest-grazing during the soil thawing periods (ST1 and ST2) alleviated microbial C and N limitations compared to the control. Mantel tests and random forest analysis demonstrated that soil temperature, soil infiltration rate, and available nitrogen content were the primary factors influencing microbial C and N limitations. These findings provide data support for determining optimal resting grazing periods in cold-season pastures of the eastern Qilian Mountains.