To date, knowledge associated with extraction, chemical constituents and bioactivities of Total Flavonoids from Carex meyeriana Kunth (TFCMK) that remains unclear. Therefore, this paper was designed to optimize the ethanol reflux extraction method by response surface methodology and determine the chemical constituents by Liquid Phase-Mass Spectrometry (HPLC-MS); additionally, the bioactivities including antioxidant, antibacterial and hemolysis test were also explored. The maximum TFCMK extraction yield of 6.248%±0.016% was obtained under the optimal conditions as follows: Ethanol concentration 50%, ratio of liquidto-solid 31 mL/g, extraction time 91 min and extraction temperature 71°C. Meanwhile, 9 compounds of TFCMK were identified by using LP-MS for the first time. In addition, TFCMK showed higher antioxidant activity compared with vitamin C, as evidenced by the lower EC 50 or IC 50 of TFCMK (reducing power assay: 0.125±0.025 mg/mL, DPPH radicals: 0.28± 0.031, ABTS + radicals:0.012 ± 0.005, hydroxyl radicals: 0.028±0.008 mg/mL; The EC 50 or IC 50 of VC 0.156±0.012, 0.059±0.019, 0.068±0.024 and 0.032±0.007 mg/mL, respectively.). Broth microdilution assays had demonstrated that the minimal inhibitory concentrations (MICs) of TFCMK on Bacillus pumilus, Bacillus subtilis and Escherichia coli were 0.625±0.0032, 1.250±0.0216 and 5.000±0.0205 mg/mL, respectively. Besides, the hemolysis rate (less than 5%) in vitro implied that TFCMK possessed a good blood compatibility. In summary, this paper provides evidences that TFCMK might be applied as an antioxidant and antibacterial agent in the pharmaceutical and chemical industries.