A new polydopamine-silver nanoparticle-polydopamine-gold (PDA-AgNPs-PDA-Au) film based surface plasmon resonance (SPR) biosensor for horse IgG detection is reported. The biosensor was constructed via layer-by-layer assembly and built on Au-film. Ag ion was reduced in situ to AgNPs in the presence of PDA. The top PDA layer was used to prevent AgNPs from being oxidized and connect with antibody directly via Schiff alkali reaction. The morphology and thickness of the modified gold film were characterized using scanning electron microscope and Talystep. The experimental results showed that the PDA-AgNPs-PDA-Au film sensing platform is stable, regenerative, and sensitive for horse IgG detection. The detection limit of horse IgG obtained with the present biosensor is 0.625 μg mL -1 , which is 2-fold and 4-fold lower than that obtained with biosensor based on PDA modified Au film and conventional biosensor based on MPA, respectively. Furthermore, when challenged to real serum samples, the sensor exhibited excellent specificity to horse IgG, indicating its potential for industrial application. In this study, the researchers used non-toxic polydopamine for more effective immobilization of the antibody onto the sensing platform, instead of conventional chemical reagents. AgNPs were used as a signal-enhancing label, and the antibody was immobilized via Schiff alkali reaction directly. The new PDA-AgNPs-PDA-Au film-based SPR biosensor exhibited 4-fold and 2-fold lower detection limit compared with that of the PDA modified Au film-based biosensor and MPA-based conventional SPR biosensor, respectively. Furthermore, when challenged with chicken serum samples spiked with horse IgG, the sensor exhibited acceptable recoveries. The new sensor is remarkably stable and regenerative. The findings of this study show the potential of this new SPR biosensor to be used for practical purposes. However, further investigation is required to build sensor chips based on this new sensor for industrial application.