Electrophoretically deposited niobium oxide nanorods have been utilized to fabricate a high performance biosensor for specific cholesterol detection. The orthorhombic structure of the Nb₂O₅ platform has resulted in the stable and excellent characteristics of the fabricated biosensor. This biosensor has been able to detect cholesterol selectively in a wide detection range [25–500 mg dl⁻¹] with a sensitivity of 0.267 μA/mg dl⁻¹ cm⁻² , has a re-usability of 10 times and stability for 6 months, due to the immobilization of the bienzyme onto the matrix, assisted by the intrusion in the Nb₂O₅ nanorods. The low Michaelis–Menten constant (0.07 mg dl⁻¹) for the biosensor indicates a higher affinity of the bienzyme towards cholesterol. The sensor has also been validated with clinical samples.