The use of polydiacetylene (PDA) vesicles in sensing systems are widespread due to the interesting optical properties of this stimuli-responsive material; however, agglutination based sensing with PDA have been relatively underutilized. To demonstrate the means for rapidly generating an agglutination probe based on peptide-displaying polydiacetylene vesicles, we implement here the use of a biotin mimetic peptide functionalized to a diacetylene amphiphile for proof-of-concept detection of a multivalent target, specifically streptavidin. Tuning of the vesicle composition revealed a distinct limit in the surface density of peptide amphiphile that could be displayed for this particular peptide sequence. A wide operational detection range was demonstrated, and the result also revealed an effective agglutination response of the PDA-based probe to streptavidin suggesting possible use of future formulations in profiling other multivalent targets.