An affinity chromatography column for isolation and purification of metallothionein (MT) was prepared with Chelating Sepharose Fast Flow gel bound with bivalent copper. Zinc-induced rabbit liver, or cadmium-induced mouse liver was homogenized and precipitated with ethanol. The sample was applied on the column and equilibrated with pH4.0 acetic acid buffer. Then pH5.2 of different concentration acetic acid buffers were used for elution of MT. Two eluted peaks were obtained and identified as MT-2 and MT-1. Comparing with the traditional method——gel filtration and ion exchange chromatography, this method is simple and time-saving in laboratory-scale.