Nano-sized Au and Pt bimetallic nanoparticles loaded metal-organic frameworks (Au-Pt@UiO-66) have been synthesized at ambient conditions, and they were being utilized in an electrochemiluminescence (ECL) biosensor for protein kinase A (PKA) activity analysis and relevant inhibitor screening based on their catalytic properties. After being phosphorylated by PKA in the presence of ATP, Au-Pt@UiO-66 probes were specifically chelated to the modified electrode by the Zr-O-P bonds formed between the surface defects of UiO-66 and the phosphorylate groups of phosphorylated kemptide. The bimetallic Au and Pt nanoparticles catalyzed the luminol-H2O2 ECL reaction by accelerating the decomposition of H2O2 to produce OH● and O2● -, which could activate the luminol ions. Light was emitted when the activated luminol ions returned to their ground state, and then utilized for PKA activity detection. Due to the high synergistic catalysis activities of bimetal Pt and Au nanoparticles to the luminol-H2O2 reaction, the Au-Pt@UiO-66 probes greatly enhanced the ECL signal of luminol and offered a high sensitive ECL strategy for PKA activity analysis. In addition, the ECL intensity of the ECL system was significantly amplified, for the UiO-66 with large surface area and high porosities accommodated bimetallic Pt and Au nanoparticles which not only can provide multiple catalytic centers toward the luminol-H2O2 reaction but also promoted the electron transfer on the electrode interface. Moreover, UiO-66 could also prevent the nanoparticles from aggregating during the catalytic reactions, thus the catalytic efficiency and stability of the biosensor was further improved. Under the optimized conditions, the detection limit for PKA activity was 0.009 UmL-1 (S/N=3). Finally, the ECL biosensor was successfully applied in inhibitor screening and cell lysates PKA activity analysis, showing great promise in kinase related research. More importantly, this strategy provides a new opportunity to promote the application of Au-Pt@UiO-66 in electrochemical related sensors.