EB1 is a conserved protein that plays a central role in regulating microtubule dynamics and organisation. It binds directly to microtubule plus ends and recruits other plus end localising proteins. Most EB1-binding proteins contain an SxIP (Ser-any residue-Ile-Pro) motif. Here we describe the isolation of peptide aptamers with optimised versions of this motif by screening for interaction with the Drosophila EB1 protein. The use of small peptide aptamers to competitively inhibit protein interaction and function is becoming increasingly recognised as a powerful technique. We show that SxIP aptamers can bind microtubule plus ends in cells and can functionally act to displace interacting proteins by competitive binding. Their expression in developing flies can interfere with microtubules, altering their dynamics. We have also identified aptamers binding to human EB1 and EB3, which revealed sequence requirements similar to but distinct from each other and from Drosophila EB1. This suggests that EB1 paralogues within one species may interact with overlapping but distinct sets of proteins in cells.