A sensitive, novel and rapid chemiluminescence (CL) method combined with high-performance liquid chromatography (HPLC) separation for the determination of salicylic acid (SA) is described in this work. The method was based on the fact that SA could significantly enhance the CL of the reaction of cerium sulfate and the tris(2,2-bipyridyl) ruthenium(II) CL system in the presence of acid. Under the optimal conditions, the CL intensity was linear over concentrations of SA in the range of 0.02-10 × 10(-6) g/mL, with a detection limit of 8 × 10(-9) g/mL (S/N = 3). Also, the relative standard detection was 2.2% for 1.0 × 10(-7) g/mL (n = 11). The proposed method has been successfully applied to the analysis of SA in human serum samples and urine samples with satisfactory results.