Recently, reflectometry interference technology has been widely used in detections of biomolecular interaction, food security and drug screening due to its advantages in label-free and dynamic detection. Protein microarray, providing an opportunity to monitor multiple biomolecular interactions simultaneously, has a potential to be used in clinical diagnosis. In this study, we have designed and constructed a label-free, real-time reflectometry interference imaging system that detects interactions of proteins according to optical phase differences from the accumulation of biological material on solid substrates, which can perform the measurement of the change of thickness in nm level. The specific binding between human IgG and anti-human IgG is performed and continuously detected by using this label-free and real-time interference imaging platform. The thickness of SiO2 is optimized so that the interference image with high signal contrast is achieved. In addition, streptavidin coupled microsphere solution is introduced to to enlarge detection signal of the biotin-linked antibody. The experimental results show that the microsphere efficiently increases the intensity of binding sites and improves the sensitivity of detection.