Enzymes in living cells are highly dynamic but at the same time regularly confined for achieving efficient metabolism. Inspired by this phenomenon, we have prepared a novel biocatalytic membrane with high enzyme activity and stability by tuning the confinement strength of the membrane to enzyme, which was achieved via modifying the support layer of a polymeric nanofiltration (NF) membrane and reversely filtrating enzyme. A mussel-inspired coating was used to modify the support interior of the NF membrane to enhance charge and steric effects on enzyme, thus stabilizing enzyme in the membrane with little increment in mass transfer resistance for substrate and products (only 20% permeability loss with a high enzyme loading of 1.34 mg/cm2). A suitable confinement strength of the membrane to enzyme could delay the enzyme leakage and endow enzyme with certain mobility for efficient reaction. Thus, the obtained biocatalytic membrane exhibited a negligible decline in BPA removal efficiency for 7 reuse cycles (<3.5%) or 36 h continuous operation (<1%) in flow through mode, resulting in a long-term stability adequate for micropollutant removal. For the first time, enzyme mobility was defined and calculated to quantify the confinement strength of the membrane, which could be used to optimize the microenvironment for enzyme immobilization and predict the performance of the biocatalytic membrane. This work concluded that rationally regulating the enzyme mobility in the membrane and a periodic back-flushing operation for redistribution of enzymes could achieve a long-term stable removal of micropollutant in water by a biocatalytic membrane.