A specific and sensitive chemiluminescent immunoassay (CLIA) was developed for quantification of a major vitellogenin (Vg) subtype (B-type Vg; VgB) in the serum of red lip mullet (Liza haematocheila). The mullet VgB CLIA was performed using twosite method, with subtypespecific antiserum developed for purified red lip mullet VgB (a-VgB). Assay conditions were optimized with regard to antibody concentration, as well as incubation time, resulting in the typical assay range from 3.91 to 500 ng/mL. Dilution of vitellogenic female mullet serum and the serum from estrogentreated juvenile mullet appeared to be parallel to purified mullet Vg, while dilution of male mullet serum hardly revealed any positive immunoreactivity in the developed CLIA. Serum levels of VgB were quantified in 10 individuals of red lip mullet reared in an aquaculture center in Tianjin City, China. In females (n=9), production of VgB was evident but varied in their serum, the levels were ranged from 3.0 to 2 700.1 μg/mL, showing a typical trend of increase during the ovarian growth. In contract, serum level of VgB in a matured male mullet was extremely low (2.7 μg/mL), indicating no sign of estrogenic activities in this environment. In addition, no trace of gonadal abnormality was evident when histological observation was performed for all individuals. The present study provided a new tool for the quantification of the major estrogeninducible biomarker (i.e., VgB) in red lip mullet, which appeared to be better in the sensitivity in comparison with our previous assay and thus enabled us to evaluate a variety level of estrogenic activities in aquatic environment. Basic information such as typical, albeit preliminary, reproductive changes in circulating VgB levels were also provided and will endow to set a “normal” baseline, which is necessary to be established prior to interpreting “abnormal” inductions of this biomarker.