Antimicrobial peptides (AMPs) are a broad prospect for clinical application against bacterial infections of biomaterials. However, a bottleneck exists as there is a lack of simple technology to prepare AMPs on biomaterials with sufficient activity, as the activity of AMP is dependent on the correct orientation on the biomaterial. In the present study, based on the conventional AMP (Tet213: KRWWKWWRRC) and surface binding peptide (SKHKGGKHKGGKHKG), we designed an Anchor-AMP that could be directly assembled onto the surface of the biomaterial and also showed excellent antimicrobial activity. By characterizing the surface using a quartz crystal microbalance with dissipation (QCM-D), contact angle, atom force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS), we found that Anchor-AMP could adsorb onto the titanium surface with a strong affinity. Different from Tet213 peptide, Anchor-AMP exhibits excellent antimicrobial activity on the titanium surface being able to inhibit 95.33% of Escherichia coli and 96.67% of Staphylococcus aureus after 2.5 h. The improved antimicrobial activity is a result of improved orientation of Anchor-AMP on the biomaterial compared to that of the Tet213 peptide. In addition, the antimicrobial activity of Anchor-AMP was active for more than 24 h. The CCK-8 assay illustrated that the modified titanium surface showed negligible cytotoxicity to bone marrow mesenchymal stem cells. The in vivo results showed that it exhibited excellent antimicrobial activity after 5 and 7 days, inhibiting 89.32% and 99.78% of S. aureus, respectively. We also demonstrated that Anchor-AMP could be applied on a variety of surfaces including gold (Au), polymethyl methacrylate (PMMA) and hydroxyapatite (HA) with strong affinity and good antimicrobial activity.