To study the ultrasensitive enzymatically amplified time-resolved fluorescenceassay (TrF ) of bioactive matter, the experimental condition of trivalent terbium ion [Tb (Ⅲ) ], 5-fluorosalicyl phosphate ester (5-FSAP) and alkaline phosphatase (AP) have been investigated. It is found that there are some quantitative relationships between them. These results supply some good information for development of TrF.