Multifunctional graphene magnetic nanosheet decorated with chitosan (GMCS) is demonstrated as a promising biosensor for fluorescence spectroscopy and it can be also applied for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for sensitive pathogenic bacteria detection. Graphene-magnetic@chitosan (GMCS) can be applied for multiple applications such as fluorescence biosensor, fluorescence background suppressor, acting as co-matrix, enriching nanoprobe, and separation by external magnets for pathogenic bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) present in aqueous suspension or blood colloids. GMCS has been prepared and characterized using various techniques including TEM, UV, FTIR, XRD, Raman and fluorescence spectroscopy. Due to the non-covalent interactions among the pathogenic bacteria with the GMCS, fluorescence enhancement takes place as a function of bacterial cell number with a low number of cell detection (4.5 × 102 to 5.0 × 102 cfu mL−1) and wide linear dynamic range which indicates that the GMCS approach is an excellent quantitative methodology and highly sensitive approach for detection of bacteria cells. Graphene works intrinsically as a fluorescence quencher for blood fluorophores and cell autofluorescence as graphene typically has a long life time, which allows detection of the bacteria signals by direct fluorescence measurements. Because of the high fluorescence enhancement efficiency, and large surface area (to volume ratio) of graphene, GMCS exhibits extraordinarily high sensitivity for complex (blood) sample analysis. GMCS also can be applied as an efficient separation and preconcentration nanoprobe for surface assisted laser desorption/ionization (SALDI) and enhances the ionization of bacterial biomolecules during MALDI-MS analysis. The bacterial cell suspension was concentrated from 1 mL to 10 μL via an external magnetic field, thus it can enhance bacteria detection for MALDI-MS analysis. GMCS is an outstanding approach which is a rapid, sensitive, culture free technique and low cost biosensor for pathogenic bacteria detection.