The dynamic assembly of the Synaptic-soluble N-ethylmaleimide-sensitive factor Attachment REceptor (SNARE) complex is crucial to the membrane fusion. Traditional ensemble studies lack the information of dynamic assembly of the protein complex. Here, we apply minute force on a tethered protein complex through dual-trap optical tweezers and study the folding dynamics of SNARE complex under mechanical force regulated by complexin-1 (CpxI). We reconstructed the clamp and facilitate functions of CpxI in vitro. Specially, the 1–83 amino acids (aa) of CpxI mainly implement the facilitate function, while the N-terminal domain (NTD) plays a dominant role. CTD is mainly related to Clamping, and separate 1-83aa and C-terminal domain (CTD) of CpxI can efficiently reconstitute the inhibitory signal identical to that the full-length CpxI functions. Our observation identifies the important chaperone role of the CpxI molecule in the dynamic assembly of SNARE complex under mechanical tension, and elucidates the specific function of each fragment of CpxI molecules in the chaperone process. Additionally, the developed technique may also be applied to study the mechanobiology and folding dynamics of single molecular protein complex.