Abstract: The 4.0 generation dendrimer (4.0G-D)-porphyrin (P) dual luminescence molecule (4.0G-D-P) is brought forward as a new phosphorescence label reagent. The product (4.0G-D-P-Con A) of 4.0G-D-P labeling concanavalin A (Con A) can emit strong and stable room temperature phosphorescence (RTP) signal on the surface of polyamide membrane (PAM)，and it was also found that there is a specific affinity adsorptive (AA) reaction between 4.0G-D-P-Con A and alpha-fetoprotein variant (AFP-V). The product of the reaction can keep very well the RTP characteristics of 4.0G-D-P, and the ΔI_pwas linear correlation to the content of AFP-V. When adding Tween-80, the ΔI_pwas increase. According to the facts above, a new method of affinity adsorption solid substrate-room temperature phosphorimetry for determination of AFP-V in human serum was established basing on Con A labeled with tween-80-4.0G-D-P dual luminescence molecule. The detection limits of this method were 0.31 pg/mL with 4.0G-D and 0.43 pg/mL with P for direct assay. Determination of AFP-V in human serum with 4.0G-D or P was as well as enzyme-linked immunosorbent assay (ELISA), the sensitivity was obviously improved and the applicability was wider.