Polo-like kinase 2 (Plk2) is a potential target for the treatment of cancer, which displays an important role in tumor cell proliferation and survival. In this report, according to the analysis of critical amino acid residue differences among Plk1, Plk2 and Plk3, and structure-based drug design strategies, two novel series of selective Plk2 inhibitors based on tetrahydropteridin chemical scaffold were designed and synthesized to target two specific residues, Lys86 and Tyr161 of Plk2. All compounds were evaluated for their inhibitory activity against Plk1-Plk3 and the cellular inhibition activity on six different human cancer cell lines. All efforts led to the identification of the most potent compounds C2 (3.40 nM against Plk2) and C21 (4.88 nM against Plk2) from the first and second series of selective Plk2 inhibitors respectively. Additionally, the selectivity of C21 over Plk1/3 was significantly increased with the selectivity indexes of 12.57 and 910.06. Moreover, most of our compounds exhibited antitumor activity in the nanomolar range in the MTT assay, indicating that our compounds, especially C2 and C21 could be promising Plk2 inhibitors for further anticancer research.