The invention relates to the field of biotechnology, in particular to a phytase mutant, a preparation method and application thereof, a DNA molecule for encoding the phytase mutant, a carrier and a host cell. The mutant provided by the invention comprises a substitution of an amino acid at at least one position selected from the group consisting of 67, 72, 79, 115, 121, 295, 300, 307, 318, 329, 360, 376 and 385. The heat resistance of the mutant is remarkably improved, so that the phytase can be widely applied to feeds.