Enzyme-catalytic fluorescence determination of artemisinin (qinghaosu, QHS) was developed using pyronine B (PB) as substrate of horseradish peroxidase (HRP). The interaction between HRP and QHS was an enzyme-substrate model. The catalytic characteristic of HRP in the oxidation reaction, in which the fluorescence of PB was decreased in the presence of QHS, was studied. The steady-state catalytic rate depended upon enzyme and substrate concentrations, and the Michaelis-Menten parameters Km, Vmax and Kcat were 8.4×10-5 mol·L-1, 7.4×10-6 mol·L-1 s-1 and 50.23 s-1. The catalytic activity of enzyme was inhibited in the presence of deactivated agents and at high temperature, respectively. Under optimum conditions, linear relationship between fluorescence intensity change (F0-F) of pyronine B and concentration of QHS was in the range of 1.41×10-7―1.27×10-6 mol·L-1. The detection limit (3σ) was determined to be 2.7×10-8 mol·L-1. The proposed method was applied to the concentration determination of QHS in the media of plasma or urine samples.