The muropeptides was the fractions of peptidoglycan from cell wall after digested by mutanolysin. It could induce the spore germination. In this paper， the muropeptides were prepared from the cell wall of Bacillus. subtilis 168 by the method of enzymatic hydrolysis. Then， the high-performance liquid was used to separate and purified the muropeptides. In addition， the structure of muropeptides was identified by liquid chromatography-mass spectrometry， Fourier transform infrared spectroscopy and nuclear magnetic resonance， and the effect of muropeptides on spore germination was further studied. The results showed that the smallest unit of muropeptides obtained after separation and purification was disaccharide tripeptide， and it could induce about 90% spore to germinate.