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Matrix metalloproteinases (MMPs) are attractive biomarkers for cancer diagnosis and treatment, while it is still a challenge to precise analysis of MMP activities owing to their very low abundance in the biological samples, especially at the early stages of tumors. Herein, a peptide microarray-based metal-enhanced fluorescence assay (PMMEFA) is proposed to simultaneously detect MMP-1, -2, -3, -7, -9, and -13 activities. The assay involves immobilization of Förster resonance energy transfer dye pair decorated peptides (FRET-peptides) on a poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) coated gold nanorod modified glass slide (GNR@P(GMA-HEMA)). To fabricate the GNR@P(GMA-HEMA) slide, GNRs are self-assembled onto an aminated glass slide, and a polymer brush (P(GMA-HEMA)) is grown through a surface-initiated atom transfer radical polymerization reaction (SI-ATRP). Upon the addition of MMPs, the FRET pairs are broken due to the specific cleavage of FRET-peptides by enzymes, resulting in the recovery of fluorescence signals and further enhancement by the MEF of GNRs. The fluorescence recovery degree provides a direct indicator for MMP activity. The PMMEFA exhibits excellent sensitivity, which enables to detect MMP-1, -2, -3, -7, -9, and -13 activities, with low limits of detection (LODs) of 1.7 fg mL−1, 0.3 fg mL−1, 2.0 fg mL−1, 1.8 fg mL−1, 2.2 fg mL−1 and 14.0 fg mL−1, respectively. To substantiate the practicability of PMMEFA, MMP activities were measured in a range of matrices, encompassing cell culture medium, serum, and tumor tissue homogenate, and MMP activities can be detected only in 0.15 μL serum and 0.025 mg tumor tissue. [ABSTRACT FROM AUTHOR]