The aim of this work was to study the CO₂ bio-sequestration application of indigenous Citrobacter species and its carbonic anhydrase (CA). Intracellular CA was purified from Citrobacter freundii (CF; accession no: MH283871) isolated from limestone rock site in Kumaun region of Indian Himalaya studied for the sequestration of carbon dioxide and the formation of calcite. CF showed maximum CA enzyme activity at 11.3 EU/ml at pH 7.0 and 37 °C. Hydration of CO₂ into carbonate was characterized by calcite phase of calcium carbonate using absorption spectroscopy and imaging technique. Purified CA showed a significantly high CO₂ sequestration capacity of 230 mg CaCO₃/mg of purified as compared to crude enzyme (50 mg CaCO₃/ml of enzyme)