Recently, fluorescent macromolecules with AIE effect have attracted considerable attentions due to their remarkable optical properties. In particular, designing novel tetraphenylethylene (TPE)-based bioconjugates to construct various self-assembled nanostructures and to expand the applications have aroused great interests. Herein, we report the self-assembly of TPE-based amphiphilic glycopolypeptide bioconjugate for bioimaging and tracing of live cells. The resultant amphiphilic fluorescent glycopolypeptide P1tM-TPE could self-assemble into different nanostructures, including vesicles, spindles and porous nanosheets, which mainly depends on the water fraction in DMSO/water mixture. At the same time, the vesicles can transform to spindles when increasing the water fractions. Both the vesicles and spindles are prone to be effectively internalized by macrophages, and all of them performed outstanding intracellular fluorescent retention properties. As far as we know, this is the first report on self-assembly and applications of glycopolypeptide-TPE bioconjugate, which will deepen our understanding on the self-assembly mechanism of TPE-based bioconjugates and provide a new way for fabricating functional fluorescent materials to monitor various biological processes.