Abstract The difficulty of dispersing nanofillers and the insufficient of flexibility remains major obstacle to the expansion of nanocomposite applications. In this study, silk fibroin (SF) based nanocomposite materials were obtained using glycerol plasticized SF as the matrix and short polymer nanofibers as the reinforcing phase. Highly flexible SF films with silk I structure were fabricated by using glycerol as a plasticizer. More importantly, plasticizer glycerol significantly improved the homogeneous dispersion of short PVA-co-PE nanofibers in SF films through interfacial interactions, forming flexible and robust nanofiber-reinforced composite films. FTIR and XRD results showed that no significant change was found on secondary structure and crystal structure of SF protein after adding PVA-co-PE nanofibers. It was found that nanofiber content showed significant influence on the mechanical behavior of nanocomposite films, where the addition of 5–10% short PVA-co-PE nanofibers is preferred for achieving enhanced flexibility and strength. This study provides new options for the fabrication of robust and flexible SF nanocomposites for functional applications.