A highly sensitive electrochemical biosensor was built for the detection of kinase activity based on the DNA induced gold nanoparticles (AuNPs) polymeric network block signal amplification. In this strategy, the DNA1 conjugated AuNPs were integrated with the phosphorylated peptide by Zr(4+) and assembled into DNA-AuNPs polymeric network block by the hybridization of cDNA with each side sequences of DNA1 and joint DNA2. The kinase activity was determined by the amperometric responses of [Ru(NH3)6](3+) absorbed on the network block by electrostatic interaction. Due to its excellent electroactivity and high accommodation of the DNA-AuNPs polymeric network block for [Ru(NH3)6](3+), the current signal was significantly amplified, affording a highly sensitive electrochemical analysis of kinase activity. The as-proposed biosensor presents a low detection limit of 0.03 U mL(-1) for protein kinase A (PKA) activity, wide linear range (from 0.03 to 40 U mL(-1)), and excellent stability even in cell lysates and serum samples. This biosensor can also be applied for quantitative kinase inhibitor screening. Finally, the PKA activities from BE4S-2B, A549, and MCF-7 cell lysates were further analyzed, which provided a valuable strategy in developing a high-throughput assay of in vitro kinase activity and inhibitor screening for clinic diagnostics and therapeutics.