The present invention relates to a method of purifying native ubiquitin without acid or heat treatment. The method presented here is designed to overcome limitations of acid or heat based ubiquitin purification in two different points of view. First, it decreases a chance of mixing other proteins resistant to acids or heat. Second, it includes no harsh condition, which might denature the ubiquitin. As a result, the purification of native ubiquitin becomes possible. The ubiquitin obtained herein is expected to be used for various purposes in technical fields.