Objective To study the determination of biological activity of ?_1-AT with chromogenic substrate and its influential factors using fresh pooled normal human plasma as reference standard.Methods Measuring absorption value of reactions between residual trypsin and BAPNA at 405nm,after ?1-AT inhibition by excess trypsin.Fresh pooled normal human plasma was used as reference standard to calculate the biological activity of?1-antitrypsin.Results Good linear correlation was obtained when the plasma was diluted to 1/50 to 1/100.PEG4000,sucrose,S/D(Tween80/TNBP) and sodium caprylate did not influence the biological activity of ?1-AT,but?1-AT activity was increased by about 20% when the concentration of sodium citrate was above 0.125mol/L.Conclusion The experiment proved that?1-AT biological activity was determined using fresh pooled human plasma as reference standard,the method is stable and reliable.Except sodium citrate,all of the materials used in the assay did not influence the determination of?1-AT activity.