In recent years, researchers were attracted to nanomaterials components for their potential role in cancer treatment. This study aimed to develop a novel and biocompatible cobalt hydroxide (Co(OH)2) nano-flakes that is functionalized by glutamic acid (Glu) and conjugated to thiosemicarbazide (TSC) for anticancer activities against human breast cancer MCF-7 cells. Physico-chemical properties of the Co(OH)2@Glu-TSC nanomaterial are characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive X–ray (EDX) analysis, and Fourier-transform infrared (FT-IR) spectroscopy. MTT assay, flow cytometry, and caspase-3 activity analysis used for evaluating anticancer properties of the Co(OH)2@Glu-TSC nanomaterial. The MTT assay result showed cellular uptake of Co(OH)2@Glu-TSC and cell viability loss in a concentration-dependent. Results of flow cytometry and caspase-3 activity analysis indicated the stimulation of apoptosis through an increase in Caspase-3 and nucleus fragmentation. In general, our findings indicate the anticancer activities of Co(OH)2@Glu-TSC nanomaterial and so it can be considered as a new treatment for breast cancer. However, further in vivo studies are required to evaluate the accumulation of Co(OH)2@Glu-TSC nanomaterial in healthy organs, such as the liver, kidneys, brain, and testes, and potential toxic effects.