Hydrogen peroxide (H2O2) is an important molecule produced in various enzymatic reactions. It is especially important in electrochemical, enzymatic biosensors detecting electroinactive analytes, such as glucose, cholesterol, and glutamate. Thus, there is a strong need for materials that have high affinity for H2O2 oxidation or reduction as well as enable immobilization and sustain enzyme activity without any additional polymer layers. Carbon nanofibers (CNFs) directly grown on tetrahedral amorphous carbon (ta-C) are feasible candidates for this purpose as they possess a reasonably wide water window (1.8 V) and good activity for H2O2 reduction in physiological pH and contain innately large amounts of suitable functional groups for enzyme immobilization. Here we show their use in ultrafast (<0.05 s) detection of H2O2 with the limit of detection of 26 μM and sensitivity of 0.221 A M–1 cm–2. Moreover, we show that ta-C/CNF hybrids can be used directly without the mass-transfer limiting polymer layers as effective immobilization platforms for glutamate oxidase for further applications in ultrafast (<0.05 s) glutamate detection. Finally, rat glial cells cultured on CNFs grown from ta-C without any additional coatings, such as polylysine, showed good adhesion on CNFs and no signs of cytotoxicity, indicating suitability of the material for future in vivo applications. This simplified and miniaturized structure provides an extremely interesting platform for various different enzyme-based electrochemical sensors.