A gold nanoparticle-based localized surface plasmon resonance substrate has been developed as nano-sensors for various bio-applications. However, reproducible and robust sensing substrates anchored gold nanoparticles has not yet been explored. In this study, dopamine-coated gold nanorods (DGNRs) were prepared and immobilized onto the micro-grooving PDMS substrates (mgPDMS). Subsequently, HER2-specific aptamers were conjugated with DGNR/mgPDMS for ECD-HER2 detection. By screening of the optimal concentration of DGNR and aptamers, the effective HER2-specific aptasensor was built up. In particular, the real-time binding assay for the evaluation of limit-of-detection (<5 ng ml-1) was conducted. Furthermore, the binding kinetics for ECD-HER2 was investigated under the biological fluid using a rat serum. Our HER2-specific aptasensor demonstrated the effective sensitivity and selectivity for ECD-HER2.