SNAP-25 (synaptosomal-associated protein of 25 kDa) is a prototypical intrinsically disordered protein (IDP) that is unstructured by itself but forms coiled-coil helices in the SNARE complex. With high conformational heterogeneity, detailed structural dynamics of unbound SNAP-25 remain elusive. Here, we report an integrative method to probe the structural dynamics of SNAP-25 by combining replica-exchange discrete molecular dynamics (rxDMD) simulations and label-based experiments at ensemble and single-molecule levels. The rxDMD simulations systematically characterize the coil-to-molten globular transition and reconstruct structural ensemble consistent with prior ensemble experiments. Label-based experiments using Förster resonance energy transfer and double electron-electron resonance further probe the conformational dynamics of SNAP-25. Agreements between simulations and experiments under both ensemble and single-molecule conditions allow us to assign specific helix-coil transitions in SNAP-25 that occur in submillisecond timescales and potentially play a vital role in forming the SNARE complex. We expect that this integrative approach may help further our understanding of IDPs.