Carbonic anhydrase is a metalloenzyme containing zinc in its active center. In this study, the use of carbonic anhydrase enzyme in the bio-mining of Balkaya lignite coal has been matched. Carbonic anhydrase (carbonate hydrolyses: E.C. 4.2.1.1) was purified from bovine erythrocytes using a Sepharose-4B l-tyrosine sulfanilamide affinity chromatography. Hydratase and esterase activity of the enzyme were determined. The purity of the enzyme was recognized by single band on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, Balkaya lignite coal with the size −20 mesh was investigated to break into pieces by using purified enzyme carbonic anhydrase with affinity chromatography. As a result of the bio-mining experiments, the carbonic anhydrase enzyme grounded Balkaya lignite coal. It was understood that the purified enzyme made smaller 1.75-fold of the Balkaya lignite coal within 96 hours using a shaker against the blank. When using a magnetic stirrer in the experiments were shown the enzyme made a smaller 73.75-fold against the blank. The results of these experiments showed that the enzyme carbonic anhydrase can be successfully used in the bio-mining of lignite coal of the same type.