Poly(lactic-co-glycolic acid)/polyvinyl pyrrolidone + poly(ethylene oxide) [PLGA/(PVP + PEO)] scaffolds with different polymer concentrations were fabricated using multilayered electrospinning, and their physicochemical properties and biocompatibility were examined to screen for scaffolds with excellent performance in tissue engineering (TE). PLGA solution (15% w/v) was used as the bottom solution, and a mixed solution of 12% w/v PVP + PEO was applied as the surface layer solution. The mass ratios of PVP vs. PEO in each 10 ml surface layer mixed solution were 1.08 g: 0.12 g; 0.96 g: 0.24 g; and 0.84 g: 0.36 g. Compared to the conventional electrospinning method used to fabricate the pure PVP + PEO (0.96 g: 0.24 g, Group A) scaffold and pure PLGA (Group E) scaffold, the multilayer electrospinning technique of alternating sprays of the bottom layer solution and the surface layer solution was adopted to fabricate multilayer nanofiber scaffolds, including PLGA/(PVP + PEO) (1.08 g: 0.12 g, Group B), PLGA/(PVP + PEO) (0.96 g: 0.24 g, Group C), and PLGA/(PVP + PEO) (0.84 g: 0.36 g, Group D). The morphology and characteristics of the five scaffolds were analyzed, and the biocompatibilities of the cell-scaffold composites were assessed through methods including Cell Counting Kit-8 (CCK8) analysis, 4',6-diamidino-2-phenylindole (DAPI) staining, and scanning electron microscopy. Therefore, with a PVP-to-PEO mass ratio of 0.96 g: 0.24 g, an optimal multilayer nanofiber scaffold was fabricated by the multilayer electrospinning technique. The excellent biocompatibility and mechanical properties of the scaffold were confirmed by in vitro experiments, which demonstrated the scaffold's promising application potential in the field of TE.