In the present study, the influences of the spacer length of the glycolipid receptors as well as the different diacetylenic lipid matrixes in color changeable polydiacetylenic vesicles on the detection of Escherichia coli (E. coli) were studied. The experimental results demonstrated that the glycolipids with longer spacer would be benefit to detecting E. coli. As a matrix lipid for colorchangeable polymer vesicles to detect E. coli, 2,4-heneicosadiynoic acid (HCDA), which has a shorter hydrophobic chain than 2,4-tricosadiynoic acid (TCDA), was more sensitive. The color change of the polymer vesicle solution using HCDA or TCDA as matrix was rapid, changing about 70-90% within 20 s, and nearly finishing in 2 min.