A highly sensitive, amperometric l-amino acid biosensor possessing unique physical, electro-conductive properties of nanomaterials is described for determination of l-amino acids (LAAs) level in fruit juices. LAA biosensor was constructed after immobilization of l-amino acid oxidase purified from goat kidney onto a c-MWCNT/ZnONPs/PANI matrix by carbodiimide linkage. The scanning electron micrographs confirmed the immobilization of globular enzyme onto the electroploymerized c-MWCNT/ZnONPs/PANI composite. The enzyme electrode detects LAA level as low as 0.35μM at a signal to noise ratio of 3 within 4s at pH 6.5 and 35°C. The fabricated LAA biosensor showed linear working range of 0.001–70mM, a sensitivity of 46.82μAmM−1cm−2. Employment of c-MWCNT/ZnONPs/PANI composite in the present electrode leads to an increase in shelf-life of electrode system to 120 days, when stored at 4°C with slight loss of enzyme activity. Efficiency of the present amperometric LAA biosensor was validated by quantitative estimation of LAA level in different fruit juices.