Bombyx mori (B. mori) silk fibroin (SF) microcapsules have acted as a great candidate in delivering drugs. However, it is difficult to fabricate SF nanocapsules using the present layer-by-layer (LBL) technique. In addition, the current SF microcapsules have limits in loading negatively charged drugs. Here, we invent a novel LBL method by introducing silane (APTES) as a structure indicator to produce SF nanocapsules that can load drugs with negative or positive charge. LBL assembly was completed by alternately coating SF and APTES on the template of polystyrene (PS) nanospheres by electrostatic attraction. SF nanocapsules were obtained after removal of the PS templates. Zeta potential analysis proved LBL assembly was indeed driven by the interaction between negative charge of SF and positive charge of APTES. Fluorescence images and electric microscope images indicated that SF nanocapsules had a hollow and stable structure with diameter at nearly 250 nm. The highest encapsulation rate of DOX or Ce6 were up to 80% and 90%, respectively, indicating SF nanocapsules have a high loading capability for both cationic and anionic drugs. In vitro cell experiments proved the biocompatibility of SF nanocapsules and their burst drug release in response to acidic environment. Furthermore, chemotherapy and photodynamic therapy proved SF nanocapsules loaded with DOX or Ce6 had significant inhibition on tumor cells. Our results suggested that this LBL technique is a facile method for polymers with negative charge to fabricate nanocapsules for antitumor drug carrier.