The ability to deploy decentralized laboratories with autonomous and reliable disease diagnosis holds the potential to deliver accessible healthcare services for public safety. While microfluidic technologies provide precise manipulation of small fluid volumes with improved assay performance, their limited automation and versatility confine them to laboratories. Herein, we report the utility of multicolor assay-on-a-chip processed by robotic operation (MACpro), to address this unmet need. The MACpro platform comprises a robot-microfluidic interface and an eye-in-hand module that provides flexible yet stable actions to execute tasks in a programmable manner, such as the precise manipulation of the microfluidic chip along with different paths. Notably, MACpro shows improved detection performance by integrating the microbead-based antibody immobilization with enhanced target recognition and multicolor sensing via Cu2+-catalyzed plasmonic etching of gold nanorods for rapid and sensitive analyte quantification. Using interferon-gamma as an example, we demonstrate that MACpro completes a sample-to-answer immunoassay within 30 min and achieves a 10-fold broader dynamic range and a 10-fold lower detection limit compared to standard enzyme-linked immunosorbent assays (0.66 vs 5.2 pg/mL). MACpro extends the applications beyond traditional laboratories and presents an automated solution to expand diagnostic capacity in diverse settings.