L-glutamate is widely used as a flavour enhancer in various foodstuffs and seasoning in the form of monosodium glutamate (MSG). MSG has been linked to neurotoxic effects, metabolic disorders, headache, numbness and palpitation. In this work, an amperometric L-glutamate biosensor based on photocurable poly(2-hydroxylethyl methacrylate)-containing ferrocene film for the determination of L-glutamate in food samples is described. The sensor is fabricated based on simultaneous immobilization of both L-glutamate oxidase and ferrocene as a mediator during the deposition of poly(2-hydroxylethyl methacrylate) film via photocuring. Ferrocene was used to shuttle the electrons directly between the reduced enzyme and the electrode. From electrochemical studies, a linear response towards L-glutamate in the concentration range of 10-30 mM was obtained at applied potential of +0.25 V with the detection limit of 7.7 mM. The storage stability of the biosensor is up to 4 months under storage condition of 4oC in refrigerator. The performance of the biosensor was applied to the determination of L-glutamate in food stocks from local supermarkets. Results from amperometric L-glutamate biosensor were further validated with HPLC method. © 2022, Malaysian Society of Analytical Sciences. All rights reserved.