Phosphorylation serves as an important post-translational modification implicated in cellular signaling and regulation. In this work, real-time monitoring of site-specific phosphorylation of p53 protein by several protein kinases, followed by its interaction with MDM2 protein was conducted using surface plasmon resonance (SPR). The binding of phosphorylated p53 to MDM2 yields a smaller SPR signal in comparison with that in the case of unphosphorylated p53 protein. Three specific protein kinases were involved in the in situ phosphorylation of the surface-confined p53 protein, and the binding kinetics between the phosphorylated p53 and MDM2 protein was monitored. The results indicate that phosphorylation of Ser15 and Ser37 at the p53 transactivation domain 1 (TAD1) by DNA-dependent protein kinase (DNA-PK) is critical for inhibiting the p53–MDM2 interaction, and the weaker binding affinity is most likely caused by the hydrophobicity change in the vicinity of the MDM2-binding motif or phosphorylation-induced p53 conformational change. In contrast, phosphorylation of Ser46 at the p53 TAD2 domain by c-Jun NH₂-terminal kinase 2α2 (JNK2α2) exerts a weaker influence on the binding affinity, whereas phosphorylation of Ser376 and Ser378 at the C-terminus of p53 by protein kinase C (PKC) appears to have little effect. The feasibility of the method for the screening of the DNA-PK inhibitor and the inhibitor of p53–MDM2 interaction has been demonstrated and the half-maximal inhibitory concentration (IC₅₀) values of wortmannin and Nutlin-3 (21 nM and 83 nM, respectively) were highly comparable with those obtained by other methods. The proposed method holds great promise for monitoring protein phosphorylation and unraveling the post-translational modification mechanism.