Abstract: Increased serum levels of unconjugated bilirubin have been associated with the brain damage of newborns. However, there are no methods to directly determine the serum levels of unconjugated bilirubin in clinical. In this study, we cloned the UNAG from Anguilla japonica, which was then expressed in prokaryotic cell. The recombinant protein was purified by Ni²⁺-NTA, and the concentration reached 1.8 mg/cm³ with high purity confirmed by silver staining method. Based on the characters that UNAG can be conjugated with unconjugated bilirubin and glow, a method for the detection of unconjugated bilirubin was established. Linear relationship was fine between the concentration of UC-BR and the fluorescence intensity of UNAG and the regression equation was established as Y=17.96X+4.333 3 (R=0.994 9). The method can be applied for the detection of unconjugated bilirubin in clinic.