Bacillus velezensis strain S3-1 has a broad range of hosts and is used as a biocontrol agent and biofertilizer. However, the interaction of maize root exudates and colonization of the strain S3-1 has not yet been investigated. In our study, strain S3-1 effectively colonized both rhizosphere soil and root tissue. Collected maize root exudates significantly induced the chemotaxis, cluster movement, and biofilm formation of strain S3-1, showing increases of 1.43, 1.6, and 2.08 times, respectively, compared with the control. In addition, the components of root exudates (organic acids: citric acid, malic acid, and oxalic acid; amino acids: glycine, proline and phenylalanine; sugars: glucose, fructose, and sucrose) were tested. Each of these compounds could induce chemotactic response, swarming motility, and biofilm formation significantly. The strongest chemotactic response and swarming motility were found when malic acid was applied, but maximal ability of biofilm formation was stimulated by proline. Furthermore, we found that these compounds of root exudates stimulated the population of S3-1 adhering to the maize root surface, especially in the presence of malic acid. These results indicate that maize root exudates play an important role in the colonization of S3-1, and provide a deeper understanding of the interaction between plants and microorganisms.