An optical biosensing scheme based on urease encapsulated calcium alginate microspheres which are coated with polyelectrolyte nanofilms predominantly composed of cresol red (CR) dye is demonstrated in this paper. The dye molecules within the nanofilms are deposited via the layer-by-layer (LbL) self-assembly technique on the microspheres and used as the optical transducer. A flow through cell constructed using a cuvette attached to a fiber optic spectrometer was used to determine the response of the biosensor to standard urea solutions of different concentrations. The change in pH and the absorbance ratio was monitored with time and these results were used for measurements of urea concentrations in the spent dialysate fluid. The biological parameters controlling hemodialysis such as dialyzer clearance or Kt/V and percent removed urea (PRU) have also been reported. The results demonstrate that the urea biosensor is pH reversible with a sensitivity of 0.09 pH units/min and is able to detect a change of 0.005 ratio units in urea concentration ranging 0.1-60 mg dL(-1). The response time of the sensor was calculated as 8 min while the detection range of urea covered the levels that are present in the spent dialysate fluid. The results obtained in the analysis of biological samples were in good agreement with those obtained by a reference method, showing no significant differences at a confidence level of 95%.