A method was developed for the direct determination of netilmicin in rat serum using high performance liquid chromatography and resonance Rayleigh scattering (RRS). The separation was carried out on a C18 column with the isocratic elution of the mixture of 20 mmol/L sodium acetate aqueous solution and methanol containing 0.22% trifluoroacetic acid (92:8, v/v). With the molecular recognition probe of pontamine sky blue, netilmicin formed an ion-association complex and enhanced the intensity of RRS. The RRS signal was detected by a commercial fluorescence detector at 365 nm of both excitation wavelength (lambda ex) and emission wavelength (lambda em). Under the optimized conditions, the limit of detection (LOD, S/N = 3) for the netilmicin was 0.7 mg/L. With the internal standard (IS) of tobramycin, a linear calibration curve ranged from 1.2 mg/L to 30 mg/L was obtained. The presented method can be used for the pharmacokinetics study of netilmicin in rat serum, and can be a new choice for the determination of aminoglycoside antibiotics in biological samples.