[Objective] Fosmid library of cultured human dental plaque samples was constructed and screened for antibiotic resistance genes. [Methods] Dental plaques from 20 individuals were obtained and cultured anaerobically in vitro. Bacteria DNA was extracted and Fosmid library was constructed. Antibiotic resistant clones were selected by plating on LB agar containing one of the three antibiotics: kanamycin, tetracycline, and ampicillin. Inserts conferring resistance were sequenced and annotated. [Results] A metagenomic Fosmid library was generated, which contained approximately 18 480 clones. Three antibiotic resistance genes were obtained through functional screening, including one kanamycin resistance gene encoding bifunctional aminoglycoside modifying enzyme AacA-AphD, one tetracycline resistance gene tet (M) and one ampicillin resistance gene encoding class C beta-lactamase. [Conclusion] It turns out that it is possible to construct fosmid libraries using cultivated dental plaque samples to screen antibiotic resistance genes.