Hybrid micro/nanostructures composed with alternative Au nanoparticle (NP) arrays and protein dots were fabricated via layer-by-layer self-assembly and the microsphere lithography technique. These micro/nanostructures were novel protein chips which had applications in the surface-enhanced Raman spectroscopy (SERS) based immunoassay. The synthetic processes were to fabricate Au nanowell arrays initially by using the templates of ordered monolayers of polystyrene (PS) microsphere arrays. Then, the proteins of antibody (avidin) were imbedded in the Au nanowells. Lastly, the immune reaction was implemented by adding the antigen of atto 610-biotin. SERS spectra were recorded as the immunoassay readout, which showed the lowest detective concentration of 100 pg/mL. These new kind of SERS-based protein chips were easy to fabricate, inexpensive and supersensitive, and provide potential as a platform for the bioassays, forensics and biosensors.