Serine/threonine kinase PLK4, a critical regulator of centrosome duplication, is closely associated with tumorigenesis due to its role in centrosome amplification. PLK4 is overexpressed in multiple cancers and has recently emerged as a promising therapeutic target for TRIM37-amplified breast cancer. Developing safe and effective PLK4 inhibitors holds significant therapeutic potential. However, currently reported PLK4 inhibitors face challenges such as limited structural diversity and potential safety concerns. Here, we designed a series of amino-pyrimidine-based PLK4 inhibitors using a structure–activity relationship (SAR)-guided strategy. Half of these compounds exhibited potent PLK4 inhibition (IC₅₀ < 10 nM), with three compounds showed significant anti-proliferative activity against TRIM37-amplified MCF-7 cells (IC₅₀ < 1 μM). Compound 5f demonstrated more exceptional potency (PLK4 IC₅₀ = 0.8 nM; MCF-7 IC₅₀ = 0.48 μM), along with favorable plasma binding and liver microsomal stability. Further evaluation in MCF-7 cells revealed its ability to suppress clonogenic survival, induce mitotic arrest, and trigger apoptosis. These findings highlight 5f as a promising PLK4 inhibitor warranting further investigation.