Objective To investigate the conformational states of ADAMTS13 under different pH conditions. Methods Atomic force microscopy (AFM) was applied to pull ADAMTS13 molecules with two distinct pulling systems: the Biotin-Streptavidin system and the 6×His-Anti-His antibody system. The rupture forces and molecular contour lengths were analyzed. Results Under pH7.4 condition, when ADAMTS13 was pulled by Biotin-Streptavidin system, the mean molecular contour length was (30.93 ± 1.56) nm, exhibiting a bimodal frequency distribution with peak positions at (22.12 ± 0.01) and (49.57 ± 0.05) nm. When ADAMTS13 was pulled using 6×Hist-anti-His antibody system, the mean molecular contour length was (32.77 ± 0.72) nm, also showing a bimodal distribution, with a peak position at (25.73 ± 0.16) and (43.84 ± 0.63) nm, respectively. Under pH6.0 condition, when ADAMTS13 was pulled by Biotin-Streptavidin system, the mean molecular contour length increased to (47.07 ± 1.6) nm, and the frequency distribution shifted to trimodal, with peak positions at (22 ± 1.25), (55.09 ± 2.62) and (76.69 ± 3.06) nm. The conformation of ADAMTS13 was more extended at pH6.0 compared with that at pH7.4. Conclusion ADAMTS13 exists in ‘closed’ and intermediate conformational states at physiological pH7.4. However, at pH6.0, ADAMTS13 can adopt ‘closed’, intermediate, and ‘open’ conformational states. This study contributes to a further understanding of the role of ADAMTS13 in normal physiology and thrombotic thrombocytopenic purpura, providing insights for the development of novel recombination ADAMTS13 drugs.