Plant root exudate plays an important role in the process of material exchange between plant and soil. Moreover, it is a significant factor in ‘plantmicroorganisms’ interactions. The process of interaction between Bacillus amyloliquefaciensL-S60 and cucumbers was analyzed. The utilization and chemotaxis of the main components of cucumber root exudate, organic acids and amino acids, by L-S60 were studied. After establishing the interaction model between the strain and the cucumber seedlings, the coloning of root surface in hydroponic culture was studied via counting the strain by time. Furthermore, the genes related to root colonization, swarming motility and biofilm formation on translational level were analyzed by fluorescent quantitation PCR. The results showed that L-S60 could utilize the main organic acids (citric acid, oxalic acid, succinic acid and malic acid) and amino acids (glutamic acid, phenylalanine, cysteine and tryptophan) from cucumber root exudates and provide well utilization of malic acid and glutamic acid. Also, L-S60 showed the chemotaxis toward these components and could be strongly attracted to malic acid and glutamic acid. According to the CFUs, which could colonize on the root surface, and the relative quantitative expression of the related genes on translational level, L-S60showed strong colonization capacities on the cucumber root surface and the most amount of colonization was 1.02×105CFU/g. In conclusion, L-S60 showed great potential to become a biological control agent of practical applications in the future. 