The binding mode of metal ions with proteins is usually different in different systems. Yeast alcohol dehydrogenase (YADH) is a zinc containing metalloenzyme that catalyzes the fermentation reaction of alcohol to acetaldehyde. UV-Vis spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC) were used to investigate the interaction of nickel(II) with Yeast alcohol dehydrogenase. The binding of Ni(II) to Yeast alcohol dehydrogenase shows a 320 nm UV absorbance band and the enzyme conformational change is reflected in the fluorescence data. Both UV-Vis and fluorescence spectra exhibit biphasic kinetics for the binding process. The interaction of Ni(II) with Yeast alcohol dehydrogenase causes the enzyme to transform from a tetramer to a dimer. The conformational change of the Yeast alcohol dehydrogenase results in an increase in the denaturation temperature and in a molar enthalpy change during the DSC process. This study reveals a complex but deep-seated mechanism for the interaction of Ni(II) with YADH.