With the method of fluorescence polarization (FP), we screened small molecule inhibitors for PLK1 PBD to identify the lead compounds for antitumor drugs. FP led to the identification of a potent hit, F083-0063, whose inhibition rate was (99.7 ± 0.4) % at 10 μg·mL−1. The IC50 was calculated to be 1.9 ± 0.1 μmol·L−1 using Graphpad Prism 5. The effect of the compound on cells' multiplication was measured by MTT assay which showed that F083-0063 inhibited the proliferation of many tumor cell lines. Flow cytometry analysis indicated that the F083-0063 promoted cell apoptosis and induced cell G2/M arrest. Migration abilities of cells, evaluated using scratch test, increased significantly in the presence of F083-0063 with the migration rate as low as (37.6 ± 0.7) % at 20 μmol·L−1. Molecular linkage technique found F083-0063 had good affinity with PLK1 PBD. The results of Western blotting showed that the expression of cyclin-dependent proteins was increased after treatment with F083-0063. In summary, F083-0063 has an antitumor activity and is expected to be an antitumor lead compound targeting PLK1 PBD.