We report the synthesis of colloidal plasmonic microspheres by taking advantage of emulsions of polydimethylsiloxane (PDMS) in metal precursor solution. Within the emulsion, both the spontaneous formation and threedimensional (3D) assembly of metal nanoparticles take place at room temperature. The number of the nanoparticles being assembled in the microsphere is controllable according to the concentration of a metal precursor. In addition, owing to the surface charge and porosity of PDMS, positively charged and neutral molecules can be more concentrated in the plasmonic microsphere. We use this plasmonic microsphere for the detection of environmentally and biologically important molecules via surface-enhanced Raman spectroscopy, since 3D assembly of metal nanoparticles in the microsphere is size-comparable to a probed volume of incident light.