Tissue engineering is an emerging field which is widely explored on the fabrication of nanofiber membranes. The deposition of nanofiber membranes using a co-electrospinning approach often is limited by the non-identification of nanofiber distribution which causes difficulties in tailoring membrane’s properties. In this study, a co-electrospinning technique was used to fabricate polyurethane-chitosan/polyvinyl alcohol-elastin (PU-Cs/PVA-El) membranes. The main aim of the study was to identify the distribution of PU-Cs and PVA-El nanofibers across the co-electrospun membranes. The membranes were fabricated in a structure of nanofibrous mat by blending 3% of Cs into 8% of PU and 1% of El into 8% of PVA. Three measurement techniques were employed to achieve the aim of the study which were Raman spectroscopy for the chemical identification and mapping, fluorescence microscopy for the observation of PVA-based distribution and scanning electron microscopy (SEM) for the morphological assessment, prior and following an immersion procedure. The results for Raman spectroscopy showed a distribution of selected spectra region and the visualization of nanofiber’s distribution by using a false color mapping. The fluorescence microscopy differentiated the nanofibers based on the fluorescence exhibited from the PVA and PVA-El. The SEM analysis revealed the difference morphologies of nanofiber structure, before and after 3 min of membrane’s immersion in distilled water. The main matrix of PU exhibited little swelling while the main matrix of PVA was easily dissolved which aided in the identification of nanofibers. In conclusion, these results confirm that the chosen methods allow the identification and differentiation of PU-based nanofibers and PVA-based nanofibers, that is further essential for tissue engineering.