Proteolysis targeting chimera (PROTAC) technology has received widespread attention in recent years as a promising strategy for drug development. Herein, we report a series of novel Wee1 degraders, which were designed and synthesized based on PROTAC technology by linking AZD1775 with CRBN ligands through linkers of different lengths and types. All degraders could effectively and completely degrade cellular Wee1 protein in MV-4-11 cell line at IC50 concentrations. Preliminary assessments identified 42a as the most active degrader, which possessed potent antiproliferative activity and induced CRBN- and proteasome-dependent degradation of Wee1. Moreover, 42a also exhibited a time- and concentration-dependent depletion manner and inducing cell cycle arrest in G0/G1 phase and cancer cell apoptosis. More importantly, 42a showed acceptable in vitro and in vivo pharmacokinetic properties and displayed rapid and sustained Wee1 degradation ability in vivo. Taken together, these findings contribute to understanding the development of PROTACs and demonstrate that our Wee1-targeting PROTAC strategy has potential novel applications in cancer therapy.