An electrochemiluminescence (ECL) biosensor based on Au NPs enhanced ECL signal of S2O82--O-2 system has been constructed for ultrasensitive detection of protein kinase activity. In the presence of ATP-s and protein kinase A (PICA), Au NPs can be captured on the thoil-phosphorylated peptides modified electrode surface, generating an enhanced ECL emission of S2O82--O-2 system. With increasing the PKA activity, more Au NPs can be captured on the modified electrode surface, resulting in the gradually enhanced ECL intensity of S2O82--O-2 system. Based on the Au NPs mediated the ECL amplification of S2O82--O-2 system, the activity of PICA can be detected sensitively with a limit of detection of 0.0002 U/mL, which is much lower than the most sensitive method in previous reports. The good conductivity and high catalytic ability of the Au NPs are revealed to account for the enhanced sensitivity in this ECL biosensor. The ECL biosensor has been successfully used for monitoring PKA activity in biological samples and screening of protein kinase inhibition.