Abstract: The optical biosensor consisted of a special flow cell located in a conventional spectrophotometer; the inner walls of the flow cell were coated with an immobilized biocatalyst. Three flow injection manifolds are proposed for controlling the flow of sample solution through the biosensor: (i) a system in which the flow is halted while the sample plug is in the flow cell, (ii) a system in which the flow direction is changed iteratively, and (iii) an open - closed loop that permits the sample to be circulated continuously through the flow cell. The performances of these manifolds were compared by use of immobilized β-glucuronidase for the kinetic determination of 4-nitrophenyl β-D-glucuronide; the 4-nitrophenol produced was detected at 405 nm. The optimized operating parameters for each manifold are given. By comparing sensitivities, α-coefficient and sampling frequencies, it is concluded that manifold (ii) is marginally superior to manifolds (i) and (iii).