The invention discloses a device and method for calibrating an on-line chromatographic instrument of a transformer. As a main oil tank, an adjusting oil tank, an auxiliary oil tank and a transformer-oil determined-value sample output system are matched, transformer-oil standard samples with the large capacity and the any-characteristic gas concentration can be accurately prepared, on-line chromatographic calibrating of oil matched with the power transformer is convenient, various characteristic gas ingredients and the content in the oil are accurate and controllable, and constant-value storage can be achieved for a long time at the normal temperature. The prepared transformer oil can be subjected to continuous and constant-pressure outputting, use cost is low, and operation is safe, rapid and convenient. As the transformer oil is recycled and purified, economical and environment-friendly effects are achieved.