ABSTRACTIn order to eliminate membrane fouling and to ensure enzymatic hydrolysis of urea, multifunctional biocatalytic membranes were prepared by using urease (URE) and trypsin (TRY) enzymes on the sulfonated polysulfone (SPSf) ultrafiltration membrane via layer‐by‐layer (LbL) self‐assembly method. The membrane architecture consisted of multilayer assembly with TRY and URE enzymes as the outer layer and inner sandwiched layer, respectively. Polyethyleneimine (PEI) and alginate (ALG) were used as cationic and anionic polyelectrolytes. Sulfonation and PEI deposition were successfully accomplished as confirmed by attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC) and scanning electron microscopy analysis, contact angle measurements, staining with toluidine blue and Congo red dyes and dead‐end filtration experiments. A characteristic value of SPSf membrane with a high water permeability (1000 L/m2.h.bar) and 95% bovine serum albumin (BSA) rejection was observed. In static conditions, URE activities of SPSf2‐PEI‐URE membrane were not affected by BSA fouling, while TRY immobilizations with increased concentrations (SPSf2‐PEI‐URE‐PEI‐ALG‐TRY) significantly lowered the activity of URE. In dynamic conditions, each deposited layer exhibited individual resistance to flow that can be considered as irreversible fouling and caused 90% of flux decline for the SPSf2‐PEI‐URE‐PEI‐ALG‐TRY membrane assembly. The recovery of the initial flux for the multilayered membrane at the end of six fouling and washing cycles was observed 85%. Moreover, at the end of 5 cycles, 78% of the URE initial activity of the multilayered membrane was preserved. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48750.