Present work describes enzyme-based optic fiber biosensor for the estimation of inorganic phosphate. A biosensor is based on the inhibitory effect of inorganic phosphate on acid phosphatase activity. Acid phosphatase catalyzes the conversion of p-nitrophenyl phosphate to a colored product p-nitrophenol, which is detected optically at 405 nm. This reaction is inhibited by inorganic phosphate present in the sample and the extent of inhibition is correlated to its concentration. Acid phosphatase immobilized in agarose–jellose composite is used as biocomponent for the fabrication of optic fiber biosensor. A calibration curve for inorganic phosphate was obtained with KH2PO4 and used for the determination of inorganic phosphate in the sample. This optic fiber biosensor was applied for the estimation of inorganic phosphate in complex urine samples. This biosensor is easy to fabricate moreover it has shown a shelf life of more than 80 days with a linearity range from 20 to 100 µM. The response time of the sensor was found to be 20 min.