The interactions between galectin-3 and two types of peach-gum polysaccharides with different molecular weights (PGP-1 and PGP-2) were detected herein via self-assembly surface plasma resonance (SPR) imaging based on digital holography. Different concentrations of peach-gum polysaccharides and Galectin-3 were simultaneously detected on an SPR biochip prepared for detecting the concentrations. The standard curves were derived and the binding equilibrium constants of the reactions were calculated. The results show that the two types of peach-gum polysaccharides can directly bind to Galectin-3. The binding equilibrium constants of PGP-1 and PGP-2 are 8.36×10 5 and 1.24×10 5 M -, respectively. The binding curves conform to the law of biomolecular interaction, demonstrating the feasibility of the proposed method in high-throughput biological detection. The proposed method can be easily controlled and is simple, label-free, and inexpensive. It is potentially applicable to the high-throughput microanalysis technology.