In the paper, we successfully prepared spider silk fibroins (Ss)/poly( d, l-lactide) (PDLLA) composite fibrous nonwoven mats for the first time to the best of our knowledge. The morphology of the fibers was observed by a scanning electron microscope (SEM) and transmission electron microscope (TEM). The secondary structure change of the spidroin before and after electrospinning was characterized using Fourier transform infrared spectroscopy (FT-IR). Herein, a qualitative analysis of the conformational changes of the silk protein was performed by analyzing the FT-IR second-derivative spectra, from which quantitative information was obtained via the deconvolution of the amide I band. A mechanical test was carried out to investigate the tensile strength and the elongation at break. A water contact angle (CA) measurement was also performed to characterize surface properties of the fibers. The cytotoxicity of electrospun PDLLA and Ss-PDLLA nonwoven fibrous mats was evaluated based on a CCL 81(Vero) cells proliferation study. The results showed that the hydrophilic and mechanical property of the composite fiber were improved by introducing spidroin.