Many studies of drug delivery nanoplatforms have explored drug loading affinity and controlled release. The nanoplatforms can be influenced by their inherent building blocks. Natural polypeptide silk fibroin (SF) is an excellent nanoplatform material because of its high biocompatibility and unique structural properties. SF secondary structures have different properties that can be changed by external stimuli. Thus, the characterization of SF-containing platforms is strongly affected by secondary structure transformations. Structural changes can occur spontaneously, which hinders the control of structural variation in aqueous conditions. Herein, we successfully prepared a controllable secondary structure composed of SF/heparin (HEP) layer-by-layer assembled nanofilms using simple solvents (glycerol and methanol). SF in the SF/HEP nanofilms takes up than 90%, which means configurations of SF have a strong effect on the character of the nanofilms. We investigated the degradation profiles of SF/HEP nanofilms depending on their β-sheet contents and demonstrated an immediate correlation between the transformation of secondary structures inside the nanofilms and the degree of degradation of nanofilms. Finally, SF/HEP nanofilms were used as a delivery platform for incorporating the anticancer drug epirubicin (EPI). We could control the loading efficiency and release profile of EPI with various β-sheet contents of the nanofilms.