Surface plasmon resonance imaging (SPRi) offers a compelling method for high-throughput, real-time, and label-free biomolecular interaction studies and immunoassays, but its performance suffers from limited intrinsic sensitivity and low-contrast SPRi images. Herein we report a high-performance SPRi chip featuring patterned microwell array constructed by photolithography of adhesive polydopamine (PDA) thin film on conventional gold chip. The chip allows for the facile construction of region-defined sensing array on its surface with improved intrinsic SPRi sensitivity due to the intensified surface plasmon wave (SPW) in the microwells. The immunoassay performance of the as-designed SPRi chip is evaluated by using anti-ochratoxin A (anti-OTA) monoclonal antibody as a model target. The results show that this microwell array structured gold chip exhibits ca. 18%–32% higher signal intensity than the conventional gold chip when detecting anti-OTA at different concentrations, and the noise remains at the same level, showing enhanced intrinsic sensitivity. Meanwhile, this microwell-structured chip affords clear and high-contrast SPRi images with well-defined sensing areas, which greatly facilitates the extraction and quantitative analysis of detection signals while efficiently suppressing the disturbance from background areas.