After constructed a 3D-Model and make the multiple sequence alignment of amino acid sequences of trehalose synthase from Meiothermus ruber CBS-01, we performed site-directed mutagenesis of D200G/H165R, R227C, R392A. And the ablity of convertion was detected. D200G/H165R and R392A lost their activities basically, while the ability of convertion of R227C declined at 50°C. When reacted at 37°C, D200G/H165R lost its activity, while R392A and R227C dropped their ability. At last, we found that R392 and D200 had important role on activity of enzyme, while R227 had little affection.