An ascorbate oxidase (EC 220.127.116.11) purified from Lagenaria siceraria fruit was immobilized onto egg shell membrane through glutaraldehyde coupling with 73.3% retention of its initial activity and a conjugation yield of 0.097 mg/cm2. The membrane consisting of ascorbate oxidase was mounted over an Au electrode to construct a working electrode for ascorbate biosensor. The biosensor showed optimum response i.e. current in mA within 10 s at pH 6.0, 40 °C and 0.6 V using Ag/AgCl reference and Cu wire as auxiliary electrode. There was a linear relationship between l-ascorbic acid concentration in the range 1 × 10-5 M and 4 × 10-4 M and current. The biosensor was employed for determination of l- ascorbic acid in serum, fruit juices and vitamin C tablets. The analytical recovery of added ascorbate in sera was 98.2% and 96.7%. Within batch and between batch coefficients of variations (CV) in ascorbate of sera were <3.6% and <4.49% respectively. l-Ascorbate values obtained for fruit juices and vitamin C tablets by present method and by DCPIP (2,6-dichlorophenolindophenol) method, showed a good correlation (r = 0.993). The biosensor has advantages such as fast response time (10 s), good repeatability (200 assays) and long-term stability (50% of its initial sensitivity after 4 months of storage). © 2010 Elsevier B.V. All rights reserved.