This study reported an improved method of trisaccharide (GlcNAcβ1, 3Galβ1, 4Glc) synthesis by fermentation of recombinant Escherichia Coli. The first part was designed for an induction temperature study. We want to compare the level of enzyme activity at different temperature and select the best condition to repeat the induction temperature in a fermenter to improve the production yield. First, the trisaccharide was synthesized at different induction temperature by direct fermentation (4L flask) of E. coli JM109 (DE3) harboring the LgtA-pET22b plasmid. The bacterial growth and trisaccharide production were compared. Second, the best condition for bacterial growth and induction of recombinant enzyme expression was repeated in a fermenter to improve its production yield. After simple two-step purification, final products were obtained with purity above 90% and all products were characterized by MALDI-TOF and HPLC.