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The specific interaction between human Toll-like receptor 9 (TLR9)–ectodomain (ECD)–fusion protein and immunostimulatory CpG–DNA was measured using force spectroscopy. Flexible tethers were used between receptors and surface as well as between DNA and atomic force microscope tip to make efficient recognition studies possible. The molecular recognition forces detected are in the range of 50 to 150 ± 20 pN at the used force-loading rates, and the molecular interaction probability was much reduced when the receptors were blocked with free CpG–DNA. A linear increase of the unbinding force with the logarithm of the loading rate was found over the range 0.1 to 30 nN/s. This indicates a single potential barrier characterizing the energy landscape and no intermediate state for the unbinding pathway of CpG–DNA from the TLR9–ECD. Two important kinetic parameters for CpG–DNA interaction with TLR9–ECD were determined from the force-loading rate dependency: an off-rate of koff = 0.14 ± 0.10 s-1 and a binding interaction length of xβ = 0.30 ± 0.03 nm, which are consistent with literature values. Various models for the molecular interaction of this innate immune receptor binding to CpG–DNA are discussed. Copyright © 2012 John Wiley & Sons, Ltd.