Due to the exceptional biocompatibility of spider silk and poly-L-lactic acid (PLLA), electrospun PLLA or spidroin fiber mat is one of the best biomaterials. The diameter of electropun PLLA fiber decreased and mechanical property of such fiber mats was improved once some spidroin was added into PLLA/Hexaflorisopropanol (HFIP) solution. The influence of electrospinning voltage and distance on the shape and mechanical properties of spidroin/PLLA composite fibers was investigated as well as the molecular conformation and crystalline structure of the electrospun fibers. The results revealed that the diameter of spidroin/PLLA fiber was non-uniform, which varied from 300nm to 1000nm. The content of random coil or α-helix structure was about 45%, and spidroin presented a crystal structure separated with PLLA in their composite fibers. With the raise of voltage, the proportion of nano-sized fibers in an electrospun spidroin/PLLA fiber mat increased and tended to be unchanged. The breaking strength of the mat was improved following the increase of voltage. Meanwhile, the ratio of nano-sized fibers and initial modulus of the spidroin/PLLA fiber mat underwent the convex change against the electrospinning distance between the needle and collector.