The effect of the chelating agent 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) labeled hepatitis B surface antibodies(HBsAb) on time-resolved fluoroimmunoassays(TRFIA) was investigated. The labeling detection method was established. The biotin-streptavidin(BAS) system was introduced, and the multi-stage amplification effect of the BAS system was analyzed. It is found that the BCPDA-Eu3+-HBsAb-BAS fluorescent marker can emit strong fluorescence. Compared to BCPDA-Eu3+-HBsAb, BCPDA-Eu3+-HBsAb-BAS demonstrates an enhanced fluorescence intensity by over 10 times. This study provides a guidance for the next generation non-radio immunoassays in clinical diagnosis.