To address the disadvantages of poor water solubility, cardiotoxicity, and hypersensitivity reactions of paclitaxel (PTX). In this study, paclitaxel silk fibroin nanoparticles (PTX-SF-NPs) were prepared by self-assembly method, and then, the nanoparticles were encapsulated by using the outer membrane vesicles of Escherichia coli (E. coil), so that biofilm-encapsulated paclitaxel silk fibroin nanoparticles (OMV-PTX-SF-NPs) were constructed. Subsequently, the prepared nanoparticles were characterized in terms of particle size and zeta potential, and in vitro cytotoxicity studies were carried out, which showed that both PTX-SF-NPs and OMV-PTX-SF-NPs possessed good antitumor activity against tumor cells. In the in vivo biodistribution study and antitumor study, the results showed that OMV-PTX-SF-NPs could effectively increase the bioavailability of paclitaxel, prolong the action time of paclitaxel in vivo, reduce the absorption of paclitaxel in the stomach, increase the concentration of paclitaxel in tumor tissues, and significantly inhibit the growth of tumors. Overall, OMV-PTX-SF-NPs is a stable extended-release oral formulation of paclitaxel, which can effectively improve the bioavailability of paclitaxel, enhance the anti-tumor activity and reduce the adverse reactions.