In this work, aptamer modified by Cy3 (1,1’-bis(3-hydroxypropyl)-3,3,3’, 3’-tetramethylindocarbocyanine) dye which has a strong Raman signal, was applied for sensitive and specific detection of trace acetamiprid by Surface-enhanced Raman Scattering (SERS). The good stability and dispersity of negative silver colloid were obtained by adding the proper concentration of sodium polyacrylate according to the principle of colloid stabilization and coagulation. During the detection process, these high stable silver nanoparticles were needed to be agglomerated by using the agglomerating agent to form more SERS enhancement hotspots to improve the SERS intensity. The effects of different agglomerating agents (NaCl, KCl, NaOH, HNO3, H3PO4, H2SO4, HCl) were investigated using acetamiprid as a probe. The results showed that the good SERS effect was showed when the electrolyte precipitator contains H+ and PO3-4. Furthermore, UV-Vis spectra show that the surface charge properties play a decisive role in the SERS effect. Furthermore, spermine with positive charges was selected to neutralize the negative charges on the phosphoric acid skeleton of Cy3-aptamer which could shorten the distance between Cy3-aptamer and silver colloid to enhance the Raman signal. The optimum reaction times of spermine with Cy3-aptamer and Cy3-aptamer with acetamiprid were 5 and 20 min, respectively. At last, quantitative detection of the acetamiprid method was established and the linearship was established between the logarithm concentration of acetamiprid and the relative intensity of the characteristic peak area at 1 392 cm-1 divided by the OH stretching vibration of water. The linear range is from 1×10-8 to 2.5×10-7 mol·L-1. The proposed method was applied to the determination of the spiked acetamiprid in water samples with the recovery of 97.4%～99.4%. The results show that the silver colloid dispersed by sodium polyacrylate and modified by spermine were helpful to capture the Cy3-aptamer and the reactants of Cy3-aptamer with acetamiprid, that the sensitivity and reliability of the method were improved.