Centrin is a member of the EF-hand superfamily of calcium-binding proteins, which is involved in the nucleotide excision repair (NER). Reversible phosphorylation of centrin is an important regulatory mechanism in vivo and is closely related to many physiological processes. To explore the possible role of centrin in NER, the endonuclease-like activity of human centrin 2 (HsCen2) regulated by phosphorylation in the absence or presence of Tb³⁺ was investigated by spectroscopy techniques, gel electrophoresis, and molecular docking simulation in 10 mM Hepes, pH 7.4. The results showed that phosphorylation weakened the binding of Tb³⁺ to HsCen2 and enhanced the binding of DNA to HsCen2. Phosphorylation improves the endonuclease-like activity of HsCen2. In addition, Tb³⁺ is favorable for DNA binding and endonuclease-like activity of HsCen2 before and after phosphorylation. These results provide clear insights into the effects of phosphorylation on the properties of HsCen2 and offer important clues for further exploration of how phosphorylation affects protein-driven functions.