Streptococcus mutans (S. mutans) is the most common cariogenic bacteria and causes caries by forming biofilms. A novel gradient Cu-bearing titanium alloy (TC4-5Cu/TC4) was manufactured using selective laser melting (SLM) technology for dental applications, which is anticipated to inhibit the formation of biofilm. In this study, the released concentration of copper ions in both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was tested in order to assess the antibacterial property of the alloy against planktonic S. mutans, and the antibacterial and antibiofilm efficiencies of TC4-5Cu/TC4 alloy against sessile S. mutans were evaluated via quantitative antibacterial tests and biofilm determination. Reverse transcription polymerase chain reaction (RT-PCR) was performed to analyze the expression of biofilm-related genes (gtfB, gtfC, gtfD, ftf and gbpB) and acid production-related gene (ldh). The results suggested that the MIC and MBC of Cu were much higher than the release concentration of copper ions of the alloy, which was consistent with the lack of antibacterial effect against planktonic bacteria. On the contrary, TC4-5Cu/TC4 alloy exhibited significant bactericidal property against the sessile bacteria and efficient biofilm-restrained ability, and all genes detected in this research were down-regulated. The results indicated that the TC4-5Cu/TC4 alloy suppressed biofilm formation and the sessile bacterial viability by down-regulating biofilm-related genes.