As the principal microtubule-organizing center in mammalian cells, the centrosome has a central role in spindle formation and chromosome segregation during mitosis. Centrosomes contain two orthogonally arranged centrioles, which duplicate precisely once per cell cycle. Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. However, how Plk4 is regulated at subcentrosomal structures remains unknown. 3D-structured illumination microscopy image shows that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal structure analyses reveal that Cep192 N-terminal fragment (Cep192-N) and Cep152 N-terminal fragment (Cep152-N) bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152 binds to the CPB markedly better than the Cep192-N and effectively snatches the CPB away from a preformed CPB-Cep192-N complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induces defects in procentriole assembly and chromosome segregation. Thus, the sequential binding of Cep192 and Cep152 are centrally required to promote Plk4-mediated centriole biogenesis, and a failure in this process may lead to human cancer. Furthermore, based on the CPB complex structural information, Plk4 is expected as a new anticancer drug target to prevent cancer cell proliferation by controlling the CPB binding region prior to centriole duplication.