Infections associated with biomedical implants and devices pose a serious clinical challenge in hospitals worldwide. Antimicrobial peptides (AMPs) have become a great prospect to inhibit this type of infection due to their broad-spectrum antimicrobial activity and low cytotoxicity. However, it is still a challenge to apply AMPs on the biomaterial surface as the activity of AMPs is sensitive to salt or enzyme. In the present study, we prepared a spacer molecule, poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (polySBMA), on a model silicon surface via surface-initiated atom transfer radical polymerization (SI-ATRP). We then modified the antimicrobial peptide HHC36 (KRWWKWWRR) with l-propargylglycine (PraAMP) to improve its salt-tolerant activity and integrated PraAMP onto the spacer molecule using click chemistry. We employed X-ray photoelectron spectroscopy (XPS), contact angle goniometry, and atomic force microscopy (AFM) to confirm the success of the immobilization process. We also characterized the antimicrobial activity and stability of the surface with an antimicrobial assay. The results reveal that the modified surface exhibits good antimicrobial activity to inhibit 98.26% of E. coli, 83.72% of S. aureus, and 81.59% of P. aeruginosa. Furthermore, as compared to the control group without the polySBMA spacer, the modified surface improved its resistance to enzymolysis. An in vitro CCK-8 assay also illustrated that this surface showed negligible cytotoxicity to mouse bone mesenchymal stem cells.