Streptococcus mutans is the leading cause of dental caries. This bacterium causes the production of insoluble glucans from sucrose to cause the accumulation of this bacterium on the tooth surface and create a biofilm. The GlucosyltransferaseB (GtfB) gene of S. mutans seems to have responsibility for the formation and maturation of biofilm. Starch and sucrose can Increase the count of S. mutans. In this study, we assessed the influence of starch, sucrose, and starch plus sucrose on biofilm formation and gtfB gene Expression of S. mutans ATCC 35668 strain. The biofilm formation was detected by a 96-well microtiter plate crystal violet method containing Luria-Bertani (LB) broth, starch and sucrose. Gene expression of biofilm-forming (GtfB) was quantified through Real-Time PCR. The biofilm formation of S. mutans was increased to 1.54 times in 1% starch concentration, 3.30 times in 1% sucrose, and 2.26 times in the 0.5% starch plus 0.5% sucrose compared to biofilm formation in the media without sugars. The starch, sucrose, and starch plus sucrose carbohydrate also increased its expression by affecting the gtfB gene effective in colonization. These results indicate how carbohydrates directly affect gtfB gene and biofilm formation by S. mutans. Glucosyltransferase B showed great potential for diagnosis and treatment proposes in S. mutans.