Ultrathin electrospun nanofibrous membranes (NfMs) based on poly(γ-benzyl-L-glutamate) (PBLG) were prepared. Scanning electron microscopy analysis revealed the production of a high-quality, bead-free nanofibrous membrane. The membrane thicknesses, ranging from 1.7 to 4.5 μm, were found to correlate directly with membrane porosity. Raman scattering analysis was utilized to investigate the conformation of the PBLG nanofibrous membrane and to assess the effects of addition of 1 wt. % trifluoroacetic acid (TFA) into the PBLG solutions, as well as the impact of annealing at 70 °C. In addition, x-ray photoelectron spectroscopy (XPS) characterization was performed to elucidate the chemical composition of the PBLG nanofibrous membrane. The Raman and Fourier-transform infrared spectroscopy spectra indicated the characteristic α-helical conformation in both the PBLG solution and the PBLG nanofibrous membrane. Furthermore, a comparative analysis of Raman band profiles proved the absence of TFA after annealing, supporting the hypothesis of TFA evaporation post-annealing, which was subsequently confirmed by the XPS results. In addition, the results from the small punch test revealed a significant correlation between membrane thickness and stiffness, indicating that increased thickness enhances stiffness. This comprehensive study provides valuable insights into the structural and compositional properties of PBLG NfMs, laying the groundwork for future investigations into their potential applications in the field of tissue engineering.