Silk fibroin (SF) has been widely used in biomedical applications for the hydrophilicity modification of high molecular polymer materials. However, the challenge remains to immobilize SF with high structure stability and strong adhesion strength between SF and the substrate. Here, we propose an effective two-step process for modifying polyethylene terephthalate (PET) with SF: dipping PET film in SF solution and subsequently carrying out plasma-assisted deposition in SF aerosol. The structure and property analysis revealed that the SF-modified PET (PET-SF) prepared using the two-step method exhibited superior structural stability and stronger adhesion strength compared to the dip-coating method and the plasma-assisted deposition method. In addition, PET-SF prepared using the two-step method resulted in a higher concentration of SF and an increased content of active groups on its surface, enhancing its hydrophilicity compared to the other two methods. Additionally, the influence of dipping time and deposition time in the two-step method was investigated. The results demonstrated that the dipping time for 6 h and the deposition time for 3 min resulted in maximum SF grafting amount with a highly stable structure. Furthermore, the PET-SF exhibited satisfactory hydrophilicity when the deposition time was more than 3 min and showed the most hydrophilicity surface at 8 min.