A portable and magnetic SERS nanoparticle arrays platform would be developed for magnetic separation and SERS detection of environmental, water, and biomolecules samples. It is very simple, low cost, high stability, high sensitive and rapid platform with portable Raman spectroscopy to detect molecules without labelling the fluorescence dye, and the limit of detection concentration is lower than 10^-8 M.The newly developed SERS substrates were provided the enhancement of SERS intensity and detection sensitivity. The observed enhancement was explained by the existence of 3D hot junctions, particularly in the z-direction of nanosheets. In addition, the 2D SERS substrates were further immobilized Fe3O4 magnetic nanoparticles to form magnetic 2D nanoparticle sheets for magnetically separating samples, which can be potentially designed into a new medical device. Moreover, the concentrated analytes were conducted to the portable Raman spectroscopy for real-time SERS detection.The floating-typed magnetic 2D nanoparticle arrays can rapidly and magnetically separate samples, such as biomolecules (adenine), environmental water pollutants (malachite blue, heavy metal), bacteria (E. coli), virus or tumor cells. Further, the resulting samples were conducted to the portable Raman spectroscopy for real-time SERS detection. It is very simple and rapid platform with portable Raman spectroscopy to detect solution-typed samples without labelling the fluorescence dye.