A novel biotinylated and enzyme immobilized nano-bio element was first prepared by using hetero-bifunctional poly(glycidyl methacrylate-co-divinylbenzene)/polystyrene (P(GMA-DVB)/PSt) composite latex beads (Scheme). 5-(n-succinimidyloxycarbonyl) pentyl d-biotinamide (Biotin-X-NHS) was firstly reacted with the hydroxyl group on polystyrene domain of latex beads. Pyruvate kinase was then directly immobilized to the biotinylated latex beads through the epoxy groups of latex bead surface. The concentration of pyruvate was monitored spectrophotometrically, to obtain maximum velocity (V_max) and Michaelis constant (K_M) of covalently immobilized pyruvate kinase. The enzyme activity was roughly half of the free one when the concentration of substrates was 100 µM, which remained almost unchanged even after stored at 4 ˚C for 48 days.