In this work, enhanced guest-pair interactions in the cavity of cucurbit[8]uril (CB[8]) are quantitatively determined using single-molecule force spectroscopy (SMFS). Significantly, the light-driven dynamic conformational change of guest pairs leads to a rupture force switching between the connected and broken CB[8]-mediated heteroternary complexation with viologen and bis(azobenzene) derivatives. SMFS is further utilized to detect methyl viologen based on the competitive host-guest interaction toward the guest in CB[8] or CB[7]. These findings highlight the extraordinary power of SMFS in supramolecular chemistry and will contribute to the fundamental understanding of the mechanochemical behavior of host-guest interactions.