Enzymatic antibacterial finishing is an eco-friendly alternative to develop functional silk-based materials. However, the low accessibility of tyrosine residues distributed in fibroin chains restricts the laccase-mediated functionalization of silk fibers (SF). To address this issue, a highly reactive p-hydroxyphenylacetic acid-modified polyethyleneimine (mPEI) was enzymatically grafted onto fibroin using laccase, aiming at constructing an antibacterial matrix of mPEI on the fiber surface. Subsequently, in situ deposition of silver nanoparticles (i.e., AgNPs) into the newly built mPEI network was performed to form a rapid antibacterial layer. The results indicated that laccase efficiently catalyzes the mPEI coupling, the zeta potential of SF-g-mPEI increases from -32 to 21.70 mV, and the silver content reaches 1.81% after AgNP embedment. Based on the combined two-step treatments, the obtained silk fabric exhibited excellent antibacterial abilities against two bacteria, including Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The antibacterial rates of both bacteria reached 99.9% within 30 min of contact, remaining over 99.9% within 18 h of contact even after washing 10 times. The present work provides an enzyme-mediated method for construction of silk fabric with durable and rapid antibacterial activity.