The infections associated with titanium-based biomaterials have been one of the most serious postoperative complications in the orthopedic surgery. Great efforts have been made to improve the antimicrobial property of titanium-based biomaterials by virtue of the surface modification strategy. From the biomimetic perspective of vegetation roots anchoring soil, alkali treatment was conducted on metallic titanium to produce a nanoroot-structured surface in the present study; then, antimicrobial peptide was anchored within the nanoroot surface by vacuum extraction and lyophilization. As a result, the obtained antibacterial peptide-leashed titanium surface showed a hierarchical structure combining the designed nanoroot topography and the anchored antibiotic peptide. Furthermore, this modified surface could steadily release for more than 10 h in a time-dependent manner. As a consequence, the elaborate antimicrobial peptide-loaded surface demonstrated a powerful antibacterial and biofilm-resistant capability against two types of Staphylococcus, without significant cytotoxicity. Specifically, Peptide-2 can kill the most planktonic and sessile bacteria for two gram-positive bacteria. Therefore, the integration of antibacterial peptide onto titanium-based implant surface may be a hopeful tool to prevent implant-associated infections in the orthopedic surgery.