Nanogold (NG) was prepared using NaBH4 reduction of HAuCl4. The NG was modified by the tobramycin-aptamer to obtain a stable Apt-NG probe for tobramycin. The three aptamers containing 15, 21 and 27 bases were examined, and results showed that the aptamer with 21 bases was best and was chosen for use. In pH 6.8 PBS buffer solution and in the presence of NaCl, the Apt-GN probes were not aggregated. When tobramycin was added, it reacted with the Apt of Apt-NG probe to form a very stable Apt-Tbc complex and released NGs that were aggregated into big particles under the action of NaCl with three resonance Rayleigh scattering peaks at 285, 368 and 525 nm respectively. The resonance Rayleigh scattering peak increased at 368 nm due to the formation of big NG particles from the probe. The effect of pH buffer solution, its volume, and Apt-GN probe concentration on the ΔI value was considered. A 200 μL pH 6.8 PBS buffer solution and 19.1 nmol·L-3 Apt-GN, giving max ΔI value, were chosen for use. Under the chosen conditions, the increased resonance Rayleigh scattering intensity ΔI368 nm was linear with Tbc concentration in the range of 1.9～58.3 ng·mL-3, with a regress equation of ΔI=35.3ｃ-23 and a detection limit of 0.8 ng·mL-3 Tbc. A 10.0, 20.0 and 30.0 ng·mL-3 Tbc was determined five times respectively, and the relative standard deviations were 6.8%, 5.0% and 4.4%. The influence of some foreign substances was examined on the determination of 38.9 ng·mL-3 Tbc, within ±10% related error. Results showed that a 80 times of Zn2+, 40 times of L-glutamic acid, Cu2+, Mg2+ and Ca2+, 20 times of glucose and terramycin, 10 times of L-phenylalanine and glycin, 2 times of L-aspartic acid, and 6 times of bovine serum albumin (BSA) and human serum albumin (HSA) do not interfere with the RRS determination of Tbc. The results showed that this aptamer-nanogold RRS method is of good selectivity. Tbc in real sample was analyzed, and the analytical result was in agreement with that of reference results, with a relative standard deviation of 6.5%～7.6% and a recovery of 95.0%～107%.