Mechanical forces are central to most, if not all, biological processes, including cell development, immune recognition, and metastasis. Because the cellular machinery mediating mechano-sensing and force generation is dependent on the nanoscale organization and geometry of protein assemblies, a current need in the field is the development of force-sensing probes that can be customized at the nanometer-length scale. In this work, we describe a DNA origami tension sensor that maps the piconewton (pN) forces generated by living cells. As a proof-of-concept, we engineered a novel library of six-helix-bundle DNA-origami tension probes (DOTPs) with a tailorable number of tension-reporting hairpins (each with their own tunable tension response threshold) and a tunable number of cell-receptor ligands. We used single-molecule force spectroscopy to determine the probes’ tension response thresholds and used computational modeling to show that hairpin unfolding is semi-cooperative and orientation-dependent. Finally, we use our DOTP library to map the forces applied by human blood platelets during initial adhesion and activation. We find that the total tension signal exhibited by platelets on DOTP-functionalized surfaces increases with the number of ligands per DOTP, likely due to increased total ligand density, and decreases exponentially with the DOTP’s force-response threshold. This work opens the door to applications for understanding and regulating biophysical processes involving cooperativity and multivalency.