Lonicerae Japonicae Flos was one of the most widely used traditional Chinese medicine for its special biological activities. The content of luteoloside, one of its major compounds, was an important standard for the quantity control of Lonicerae Japonicae Flos. The major method used for the detection of luteoloside was instrumental analysis. Compared with the ELISA method, instrumental analysis was time-consuming, complex pretreatment and low-throughout. Thus, it was significantly important to develop an enzyme-linked immunosorbent assay (ELISA) for luteoloside analysis. Here, the conjugates of luteoloside-bovine (LG-BSA) and luteoloside-ovalbumin (LG-OVA) were produced as the immunogen and coating antigen by the carbodiimide ( CDI) method, respectively. The conjugation ratio of carrier protein and the hapten in the conjugate were determined by UV-Vis spectrophotometry (UV). LG-BSA conjugate was used to immunize Bal b/c mice to produce antiserum. The titer and specificity of antiserum were detected by ELISA. The conjugation ratio of hapten and carries protein were 3. 7: 1 (LG-BSA) and 1. 0: 1 (LG-OVA). The antiserum titer was higher than 2 000 with the linear range of 18.4-4 852.4 μg x L(-1), R2 = 0.988 4 and IC50 = 298.7 μg x L(-1). The result showed that the conjugate antigen LG-BSA was synthesized successfully and the mice can produce specific antiserum injected with artificial antigen.