Objective This study aims to explore the conformational states of ADAMTS13 under different pH conditions using atomic force microscopy (AFM) pulling techniques. Methods We applied AFM to stretch ADAMTS13 molecules with two distinct stretching 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 stretched 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) nm and (49.57 ± 0.05) nm. When ADAMTS13 was stretched 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) nm and (43.84 ± 0.63) nm, respectively. Under pH6.0 condition, when ADAMTS13 was stretched 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) nm, (55.09 ± 2.62) nm and (76.69 ± 3.06) nm. Conclusion The findings indicate that ADAMTS13 exists in "closed" and intermediate conformational states at physiological pH 7.4. However, at pH 6.0, ADAMTS13 can adopt "closed," intermediate, and "open" conformational states.