The invention relates to a synthesizing method of 4-hydroxyisoleucine. According to the method, isoleucine hydroxylase genes are screened out, recombinant expression plasmid is constructed, and fusion protein having an amino acid deaminase transmembrane domain and isoleucine hydroxylase is expressed, so that isoleucine hydroxylase is anchored to the surface of cells; then a complete cell catalysis technology is utilized to replace a cell thawing and breaking technology, a cell freezing technology is omitted, and freezing processing equipment is not needed; meanwhile, product separation difficulty caused when inclusion of the cells is spread to reaction liquid after the cells are broken is avoided, the production procedure is simplified, the energy consumption, the material consumption and the production cost are lowered by a large margin, the conversion ratio of 4-hydroxyisoleucine produced through the method reaches up to 99.0% or above, and the synthesizing method is more beneficial to industrialized production.