Receptor-ligand bond dissociation under applied force is crucial to elucidate its biological functionality when the molecular bond is usually connected to a mechanical probe. While the impact of probe stiffness, k, on bond rupture force has recently attracted more and more attention, the mechanism of how it affects the bond lifetime, however, remains unclear. Here we quantified the dissociation lifetime of selectin-ligand bond using an optical trap assay with low stiffness ranging from 3.5×10-3 to 4.7×10-2 pN/nm. Our results indicated that bond lifetime yielded distinct distributions with different probe stiffness, implying the stochastic feature of bond dissociation. It was also found that the mean lifetime varied with probe stiffness and that the catch bond nature was visualized at k■3.0×10-2 pN/nm. This work furthered the understanding of the forced dissociation of selectin-ligand bond at varied probe stiffness, which is physiologically relevant to the tethered rolling of leukocytes under blood flow. While the impact of probe stiffness, k, on bond rupture force has recently attracted more and more attention, the mechanism Here we quantified the dissociation lifetime of selectin-ligand bond using an optical trap assay with low stiffness ranging from 3.5 × 10-3 to 4.7 × 10-2 pN / nm. Our results showed that bond lifetime yielded distinct distributions with different probe stiffness, implying the stochastic feature of bond dissociation. It was also found that the mean lifetime varied with probe stiffness and that the catch bond nature was visualized at k ■ 3.0 × 10-2 pN / nm. This work furthered the understanding of the forced dissociation of selectin-ligand bond at varied probe stiffness, which is physiologically relevant to the tethered rolling of leuko cytes under blood flow.