On the basis of Fmoc solid-phase synthesis, by optimizing the degree of activation of amino acids on linear peptides and the selection of condensation reagents, the synthesis purity of linear peptide was increased to 86.3%, higher than that before optimization 28%, the separation and purification of linear peptide was omitted. For cyclic peptides with side chain protecting groups were separated by gel column instead of high performance liquid chromatography to reduce product loss and increase the total yield of cyclic peptide to 55.6%. Their structures were characterized by 1HNMR, 13CNMR and HRMS. The antimicrobial activity of Desotamide B synthesized by the above method was tested with vancomycin as the control. The results showed that the compound had a good inhibitory effect on Gram-positive bacteria, and the minimum inhibitory concentration was 16 μg / mL.