The premixing self-assembly solution of horseradish peroxide with poly(sodium 4-styrenesulfonate)(RRP +PSS) is made by blending HRP and polyelectrolyte PSS

and the active[(HRP+PSS)/PDDA]。enzyme muhilayer biofilm is fabricated by altemate deposition of (HRP+PSS) and PDDA on the surface of negative ionized PET．The topography of films

the stepwise growth of layers and the activity of HRP in the film are measured with UV-Vis and AFM．HRP and PSS are assembled on the PDDA layer together from the premixing solution of (HRP+PSS)

and the ratio of HRP to PSS in every layer of(HRP+PSS) is the same．The banded stmcture of HRP under the PSS is observed on the PDDA layer．The Michaelis Constant of the reaction of H2O2 and 4-amino-antipyrine(4-AAP) catalyzed by HRP in biofilm is 9.7×10-5mol·L-1 and is lowet than that of the reaction catalyzed by HRP in solution (1.52×10-4 mol·L-1)．The affinity between H2O2

and HRP in biofflms is rather strong．