In this article, a nanofibrous composite scaffold of poly L-lactic acid(PLA)/silk fibroin(SF)-gelatin was fabricated by multilayer electrospinning. To investigate the feasibility of PLA/SF-gelatin use as scaffolds, the porosity and mechanical properties were examined; in particular, the biocompatibilities were evaluated in vivo and in vitro by the means of cell adhesion and cytotoxicity testing, short-term subcutaneous implantation testing, and acute hemolysis testing according to the requirements of ISO 10993. The results showed the scaffold achieved the desirable levels of pliability (elastic up to 7.3% strain) and the appropriate breaking strength (2.22 MPa). The porosity of the SF-gelatin layer was 87% and the pore diameter was 142 nm. After 12 days of cultivation, SEM observation demonstrated the scaffold was nontoxic, biocompatible, and capable of supporting 3T3 mouse fibroblasts attachment, spreading, and growth. The hemolysis test proved the scaffolds with hemolytic rates from 3.1 to 4.5%. The subcutaneous implantation test indicated minor inflammatory reactions surrounding the scaffolds and biodegradation were initially observed in about 3 months. The desired porous structure, strong and pliable properties, combined with the ability of PLA/SF-gelatin scaffold to support cell growth in vitro, especially excellent biocompatibility in vivo, suggested potential application for tissue engineering scaffolds.