The planktonic-biofilm transition is reported to facilitate the survival of disease-causing pathogens in a hostile environment, as the ability of pathogens to resist antibiotic treatment is 10 to 1000 times higher in a biofilm than in the planktonic mode. The availability of nutrient components such as amino acids in the surrounding environment plays an important role in the initiation of the biofilm formation. This work proposes a metabolic modeling framework to study the influence of availability of amino acids on the biofilm formation capability of Pseudomonas aeruginosa, one of the major pathogens causing nosocomial infections. Specifically, the genes that are up-regulated during the biofilm initiation were used to determine the metabolic reactions that are positively associated with P. aeruginosa biofilm formation. A criterion was then defined from the change of fluxes of these biofilm-associated reactions and the biomass growth rates to quantify the chance of biofilm formation upon the change of amino acid uptake rates. It was found that adding one of the following eleven amino acids, including Arg, Tyr, Phe, His, Iso, Orn, Pro, Glu, Leu, Val, and Asp, into the minimal medium may trigger P. aeruginosa biofilm formation. These results are perfectly consistent with the existing experimental data. The developed modeling framework was further used as an in silico platform to investigate the impact of the availability of two amino acids on the biofilm formation. It was found that the availability of additional amino acids can enhance biofilm formation and that there may be synergistic mechanisms for multiple amino acids to promote the biofilm formation.