To improve long-term graft patient outcomes and develop more effective antirejection therapies, noninvasive monitoring of acute cellular rejection (ACR) after organ transplantation is urgently needed. As a biomarker of ACR, Granzyme B (GrB) is expected to be applied in the noninvasive monitoring of ACR. Herein, we have developed a method for detecting the GrB activity based on the target-initiated great change in electrochemical steric hindrance by designing a nanoprobe. The nanoprobe is prepared by conjugating a specific peptide, which is responsive to GrB cleavage activity, to gold nanoparticles (AuNPs). Meanwhile, a piece of DNA sequence with G-quadruplex (G4) is attached at the distal end of the peptide. Upon exposure to GrB, the peptide substrate is cleaved to eliminate the steric hindrance between inter-nanoprobes as well as nanoprobe and DNA tetrahedron (TDN), allowing the released DNA strand to hybridize with TDN, giving sensitive signal output. The method can also be used to detect GrB activity in complex biological settings, so it has a great potential for monitoring GrB activity in the blood or urine of graft patients.