In a pH=0.65-1.5 NaAc-HCI medium, methylene blue(MB) reacts with 12-tungstophosphoric acid (TPA) by virtue of electrostatic attraction and hydrophobic force to form a 3:2 ion-association complex. As a result,the intensities of resonance Rayleigh scattering(RRS), second-order scattering(SOS) and frequency doubling scattering(FDS) are enhanced greatly. The maximum scattering wavelengths of RRS, SOS and FDS are located at 316, 647and 311 nm. The increments of scattering intensity(△I) are directly proportional to the concentration of MB in a certain range. The methods exhibited high sensitivity, and the detection limits(3σ) for MB are 2.3 ng/mL(RRS method),5.6 ng/mL(SOS method) and 6.4 ng/mL(FDS method), respectively. The effects of coexisting substances have been examined, and the results indicate that the methods have good selectivity. Based on the above researches, a new spectral method for the determination of trace amounts of MB has been developed. It can be applied to the determination of MB in human serum, and the recoveries are 97.5%-105.0%. The results are in good agreement with those obtained by the pharmacopoeia method. In this work, the optimum conditions of the reaction and the influencing factors were investigated. In addition, the reaction mechanism and the reasons of the enhancement of resonance light scattering were discussed.