The residual melamine in milk is one of the important risk factors for human health. A method for the rapid quantitative detection of melamine (MEL) in milk has been designed based on surface plasmon resonance (SPR) technique. As a vector, nano-gold was used to connect MEL to the surface of SPR biosensor chip. The antibody quantity and regeneration conditions were optimized. The designed chip was tested regarding its stability and the accuracy of the proposed method was checked. Using pollution-free milk added with serial concentrations of MEL, the competitive inhibition curve was developed and used to detect real samples. The results showed that the chip exhibited a good reproducibility, with a relative standard deviation (RSD) of 5.05% for 60 replicate determinations. The detection limit of this method was 3.24 ng/mL, much lower than the maximum residue limits adopted by the Codex Alimentarius Commission and by relevant state authorities. The whole analytical procedure including sample preparation could be completed within 30 min and is suitable for rapid in-situ quantitative detection of milk.