As the three-dimensional (3D) architecture of porcine acellular dermal matrix (pADM) mimics the biological characteristics of a native extracellular matrix (ECM), it has been extensively utilized as a tissue scaffold. In this study, a novel pADM-derived micro-and nano electrospun collagen membrane (PDEC) was successfully prepared by the electrospinning technique, using porcine acellular dermal matrix (pADM) as the raw material and 1,1,1,3,3,3-hexafluoro-2-propanol (HIFP) as the solvent, and was characterized by scanning electron microscopy (SEM) analysis. In contrast, another collagen-derived electrospun collagen (CDEC) was also fabricated using pure porcine collagen as the raw material. The structure and composition of the PDEC and CDEC were measured by Fourier transform infrared (FTIR) spectroscopy, SDS-PAGE gel electrophoresis, specific intrinsic viscosity and atomic force microscopy (AFM). The results indicate that the structural integrity of the PDEC is almost maintained and only a small amount of the PDEC was destroyed into gelatin, while almost all of the CDEC was degraded. Moreover, the results of circular dichroism (CD) analysis also demonstrate that the PDEC possesses a higher content of a-helix structure but less beta-turn structure. Additionally, the results from ultrasensitive differential scanning calorimetry (US-DSC) and XRD analysis also suggest that the thermal stability and crystallization of the PDEC have little differences compared to collagen. Furthermore, the mechanical properties of the PDEC are significantly enhanced compared to those of the CDEC. Above all, the results obtained in the MTT study indicate that the PDEC has almost the same good biological activity, in terms of cytotoxicity, as natural porcine collagen, and this activity is obviously superior to that of the CDEC. In conclusion, our study provides a new pADM-derived electrospun collagen in which the triple helical structure has seldom been damaged and thus it can be applied as a novel electrospun collagen scaffold for tissue engineering.