Amphiphilic Janus Au NPs were synthesized by emulsion reaction with presence of D-penicillamine (D-PA) and benzyl mercaptan (BM) molecules at water and toluene phases, respectively. TEM and XRD characterizations indicate that the reaction product is composed of Au nanocrystals in size of 2.9 nm. XPS, FTIR, Raman and 1H NMR analyses suggest that the capping ligands on the Au NPs are D-PA and BM, where the ratio of D-PA to BM is ca. 1.7. Contact angle measurement and NOESY analysis further reveal that the hydrophilic D-PA and lipophilic BM are separated into two distinct hemispheres on the individual Au NPs. The simple and efficient approachallows large scale preparation of amphiphilic Janus Au NPs at low cost for versatile applications, such as drug delivery, vapor sensors and heterogeneous catalysts.