The invention provides site-specific mutagenesis high temperature resistant phytase gene TP and an expression vector and application thereof. Amino acid in a specific area in a phytase amino acid sequence is mutated into praline and arginine so as to enable the phytase to have a special stable protein structure. Simultaneously, a gene sequence is optimized to synthesize a phytase gene according to pichia pastoris codon preference and GC content, recombinant plasmids are built and transferred to pichia pastoris, and positive converters are obtained through screening to conduct induction expression to obtain the high temperature resistant phytase. Experiments prove that the phytase obtained through expression of the pichia pastoris can resist high temperature plasmids with the temperature higher than 85 DEG C, and survival rate reaches 85%. Popularization and application of the high temperature resistant phytase have substantial economical benefit and social benefit on development of feed animal husbandry of our country, and simultaneously great ecological benefit can be obtained.