A sensitive and selective quantum dots (QDs)-based fluorescence resonance energy transfer (FRET) biosensor was successfully fabricated for the detection of the cancer marker type IV collagenase. The cancer marker type IV collagenase could be determined by linking peptide between the donor-luminescent QDs and the acceptor-gold nanoparticles with small size (SAuNPs) based on FRET. Firstly, peptide terminated with cysteine could be bound to the surface of SAuNPs via the formation of Au–S bonds. Then QDs combined with the SAuNPs through the peptide. At this moment, the photoluminescence (PL) of the QDs was quenched. After type IV collagenase was added into the system, the SAuNPs could detach from QDs because the enzyme cleaves the peptide to lead to the disappearance of FRET, which allowed the fluorescence of the QDs to return. The enzymatic activity of type IV collagenase was related to the PL change of QDs-based FRET probes. The concentration of type IV collagenase was determined in the linear range of 0.05–10 μg mL⁻¹ with a detection limit of 18 ng mL⁻¹. This sensor opens a new route for monitoring the low activities of type IV collagenase in normal and cancerous cell cultures.