Biofilms on medical devices such as implants and catheters are one of the most urgent threats in clinic because of causing increased antimicrobial resistance. Indirect detection methods such as end-point staining methods with crystal violet in tubes or microtiter well plates for screening attached bacteria on the surfaces target to extracellular polysaccharides. These methods are simple; however, there are some limitations. First, they can only be used after the formation of the biofilms, meaning there is no chance to continue the study with the same biofilms after staining. Second, they are semi-quantitative methods and the dyes are non-specifically bound to materials and so, sensitivity and reproducibility are low. In this study, a new, rapid, quantitative, nuclease-based fluorescence method was developed for detection of the formation of biofilm. In comparison with control which is nuclease-negative bacteria, biofilms produced by S. aureus had 31 times more signal. Additionally, biofilm formed by S. aureus in media not-including H2O2 had 14 times more signal than ones with H2O2.