Abstract: In the absence of Tobramycin, the aptamer was coated on the surface of gold nanoparticles (AuNPs) to prevent salt-induced aggregation. In the presence of Tobramycin, the aptamer would separate from the surface of AuNPs and combine with Tobramycin because of the high affinity between the aptamer and Tobramycin, resulting in the aggregation of AuNPs at the appropriate content of salt, and the color of the reaction system changed from red to purple-blue. Spectrophotometry was applied to the determination of residual amount of Tobramycin in milk based on gold nanoparticles modified by aptamer. The optimized conditions found were as follows:incubation time of aptamer Hp and incubation time of Tobramycin were 15 min; pH of the reaction system was 7; concentration of aptamer Hp was 1.5 μmol·L^-1; concentration of sodium chloride was 1.5 mol·L^-1. Linearity range of Tobramycin was found between 40.0 nmol·L^-1 and 175 nmol·L^-1 with detection limit (3s/k) of 13.3 nmol·L^-1. On the base of blank sample, test for recovery was made by standard addition method; values of recovery found were in the range of 96.4%-108%, with RSDs (n=6) of determined values in the range of 1.8%-3.0%.