In this paper a rapid, selective, and ultrasensitive protocol for the detection of the active form of matrix metalloproteinase-1 (MMP-1), which is a novel predictive and prognostic biomarker, was presented, which might strengthen the current predictive systems. The biosensor construction procedure was extremely simple, economical, and time-saving, as it involved only the chemisorption step of the voltammetrically active receptor (tripeptide (Cys-Gly-Ile) labeled with methylene blue (MB) and the sealing thiol. The active form of MMP-1 was recognized based on its hydrolytic activity; as a consequence, the receptor fragment (-Ile-MB) was removed from the sensor surface. The biosensors constructed were characterized by a wide dynamic concentration response range (1.0 pg mL-1-1.0 μg mL-1) and a low detection limit (33 fg mL-1), especially the biosensor with voltammetric detection, without the amplification step. One of the important advantages of the proposed biosensors is that they can be directly used to analyze the content of the active form of MMP-1 in clinical samples without the dilution step and any other preparation step.