In this study, nanofibrous extracellular matrix (ECM) was prepared from human adipose tissue, and the stepwise products were analyzed using differential thermal analysis (DTA)/thermogravimetric analysis (TGA) and Fourier-transform infrared (FTIR)/Raman spectroscopy. Human adipose tissue was liposuctioned (sample 1), centrifuged (sample 2), pulverized, centrifuged again (sample 3), and finally freeze-dried (sample 4). Each sample was subjected to DTA/TGA and FTIR/Raman analyses. In the DTA curve of sample 1, the major peak was observed at approximately 60 °C. However, relatively flat DTA curves were detected in samples 2 and 4. In the TGA results, sample 1 showed a more rapid weight loss pattern than the other samples. Samples 1, 2, and 3 showed similar FTIR spectra with a strong, broad absorption feature at ~3400 cm-1. Amide I, II, and III bands were clearly observed in the FTIR spectrum of sample 4. Samples 1 and 2 showed typical adipose tissue Raman spectra. However, in samples 3 and 4, the Raman signal was low. Scanning electron microscopic analysis of the freeze-dried ECM (sample 4) confimed its three-dimensional porous nanofibrous structure. These findings suggest that human adipose tissue, intermediate products, and human adipose tissue-derived ECM can be easily and effectively characterized with thermal and spectroscopic analyses. These efficient analyses can aid in the preparation of ECM and in clinical applications for regenerative medicine.