Protein phosphorylation catalyzed by protein kinases plays a critical role in many intracellular processes, and detecting kinase activity is important in biochemical research and drug discovery. Herein, we developed a novel fluorescent biosensor to detect protein kinase activity based on phosphorylation-mediated assembly of semisynthetic green fluorescent protein (GFP). A chimaera S-peptide composed of the 10th β-strand of GFP (s10) and a kinase substrate peptide was synthesized. Kinase-catalyzed phosphorylation of the S-peptide can protect its s10 part against cleavage by carboxypeptidase Y (CPY). Then, the peptide can bind the truncated GFP (tGFP, GFP without s10) to assemble intact GFP and recover fluorescence. Unphosphorylated S-peptide would be degraded by CPY, and fluorescent protein assembly could not occur. Thus, the kinase-catalyzed phosphorylation can switch on the fluorescence signal. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase with a low detection limit of 0.50 mU/μL and its inhibition of H-89 with an IC50 value of 23.4 nM. The feasibility of this method has been further demonstrated by assessment of the kinase activity and inhibition in the cell lysate. Moreover, based on the reverse principle, this method was expanded to detect the activity of protein phosphatase 1. Our method, using semisynthetic GFP as a readout, is facile, sensitive, label-free, and highly versatile, thus showing great potential as a promising platform for protein kinase detection and inhibitor screening.