A graphene-based electrochemical biosensor was developed for the detection of matrix metalloproteinase 2 (MMP-2) endopeptidase, whose expression can be significantly related to the occurrence, metastasis, and prognosis of cancer. A specific anti-MMP-2 aptamer was successfully immobilized on the surface of electrochemically reduced graphene oxide via a pyrene-based linker, enabling the specific capture of MMP-2. The sensor was able to detect 1 ng mL−1, with an overall detection time of less than 20 min. Moreover, the aptamer-based biosensor showed good specificity toward different unspecific proteins.