Coffee is a favorite and beverage in Western countries that is consumed daily. In the present study, capillary zone electrophoresis (CE) was applied for the separation and quantification of three isoflavones including daidzein, genistein and formononetin in coffee. Extraction of isoflavones from the coffee sample was carried out by extraction and purification process using ether after the acid hydrolysis with the antioxidant butylated hydroxy-toluene (BHT). The experimental conditions of the CE separation method were: 20 mmol/L Na2HPO4 buffer solution, 25 kV applied voltage, 3 s hydrodynamic injection at 30 mbar, and UV detection at 254 nm. The results show that the three compounds can be tested within 10 min with a linearity of 0.5–50 µg/mL for all three compounds. The limits of detection were 0.0642, 0.134, and 0.0825 µg/mL for daidzein, formononetin and genistein, respectively. The corresponding average recovery was 99.39% (Relative Standard Detection (RSD) = 1.76%), 98.71% (RSD = 2.11%) and 97.37% (RSD = 3.74%).