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) with abilities to inhibit the formation of biofilm was manufactured using selective laser melting (SLM) technology for dental applications. In this study, the released concentration of copper ions Cu2+ in both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested in order to assess the antibacterial property of the alloy against planktonic S. mutans, and the antibacterial and anti-biofilm efficiency 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 suggest that the MIC and MBC of Cu2+ were much higher than the released concentration of Cu2+, which is consistent with the lack of antibacterial effect against planktonic bacteria. On the contrary, TC4-5Cu/TC4 alloy exhibited significantly bactericidal property against the sessile bacteria and efficiently biofilm-restrained ability, and all genes detected in this research were down-regulated. The results indicate that the TC4-5Cu/TC4 alloy suppresses biofilm formation and the sessile bacterial viability by down-regulating biofilm-related genes.