The study aimed to extract and purify the enzyme cytosine deaminase from locally manufactured bread yeast Saccharomyces cerevisia. To achieve the goal of the study, bread yeast was made locally and transported to the laboratory for an enzyme extraction process using toluene as an organic solvent for yeast wall rapturing. A crude enzyme was extracted with cold distilled water. Steps for enzyme purification started with a precipitation step with ammonium sulfate at 60% saturation, and then the ion exchange purification method ended with a gel filtration purification process applied. In each step, the supernatant volume, enzyme activity, specific enzyme activity, protein concentration, and percentage of enzyme yield were recorded. The cytotoxic effect of different enzyme concentrations in all purification steps toward the lung cancer cell line A549 and human breast cancer line MDA cell line beside normal cell line REF cells was investigated. The specific activity of the crude extract of locally produced bread yeast reached 13.205 units/mg protein, and for ammonium sulfate enzyme precipitation the specific activity amounted to 17.68 units/mg protein, for ion exchange was 216.66 units/mg protein, while for purification with gel filtration, the specific activity reached 571.428 unit/mg protein. The cytotoxic effect of the extracted enzyme in all steps; crud, ion-exchange, and gel filtration purified enzyme on the lungA549 and breast AMD cancer lines beside the REF normal cells were should a range of toxic effects at different enzyme concentrations. The toxicity was reduced till diminished as the enzyme was more purified.