We have developed a new and facile approach for synthesis, labeling, and characterizing amphiphilic nanoscale Janus particles. The aqueous dispersion of the Aerodisp W1226 fumed silica particles were embedded into polycarbonate (P.C.) microspheres surfaces via inverse solvent displacement method and further primary functionalization by an amine group. Subsequently, the polymer was dissolved, and a second modification was preferred to introduce a thiol group on the revealed embedded side of the particles, forming silica-based Janus/amphiphilic particles. We first characterized the Janus particles by laser scanning confocal microscopy and then with direct stochastic optical reconstruction microscopy (dSTORM) to detect the labeling distribution of the different functional groups. Super-resolution imaging results confirmed that the fabricated particles are Janus particles with different chemical properties on each hemisphere. The particles were also characterized by scanning and transmission electron microscopy for size and shape. The results showed aspherical particles with two hemispheres, confirming successful fabrication and chemical modification. Also, this work successfully demonstrated the synthesis, characterization and analysis of nanoscale Janus/amphiphilic particles nanoscale Janus particle.