The assembly of colloidal metal nanoparticles at immiscible liquid interface has attracted considerable attention because of both enhanced physical properties associated with the assembly and controlled mass transfer at the interface for many sensing and catalytic applications. However, rapid production of 3D assembly of metal nanoparticles at liquid interface is still challenging. Here a facile and robust method is proposed to generate 3D assembly of colloidal metal nanoparticles at oleic acid/water interface, which is based on an interesting observation of the autonomous and rapid interfacial locomotion of nanoparticles from air/water to oleic acid/water interface. This interfacial transfer of these particles is completed within a minute considerably owing to electrostatic interaction between positively charged surface molecule of the particle and the negative end of oleic acid, producing up to 20-fold denser 3D assembly. To realize one of the benefits of the proposed method, 3D assemblies of gold and silver nanoparticles at oleic acid/water interface are successfully exploited for the detection of trace molecules in both oil and water phases via surface-enhanced Raman spectroscopy. Molecular Raman signals can be increased by our 3D nanoparticle layers by up to 100 times, compared with that measured from nanoparticle monolayers being transferred onto solid substrates.