Here we report the development of a programmable and fully automatic gold array-embedded gradient microfluidic chip that integrates a gradient microfluidic device with gold-patterned microarray wells. This device provides a convenient and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay platform for cancer biomarkers. We used hollow gold nanospheres (HGNs) as SERS agents because of their highly sensitive and reproducible characteristics. The utility of this platform was demonstrated by the quantitative immunoassay of alpha-fetoprotein (AFP) model protein marker. Our proposed SERS-based immunoassay platform has many advantages over other previously reported SERS immunoassay methods. The tedious manual dilution process of repetitive pipetting and inaccurate dilution is eliminated with this process because various concentrations of biomarker are automatically generated by microfluidic gradient generators with N cascade-mixing stages. The total assay time from serial dilution to SERS detection takes less than 60 min because all of the experimental conditions for the formation and detection of immunocomplexes can be automatically controlled inside the exquisitely designed microfluidic channel. Thus, this novel SERS-based microfluidic assay technique is expected to be a powerful clinical tool for fast and sensitive cancer marker detection.