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We report the fabrication and performance of a surface plasmon resonance aluminum nanohole array refractometric biosensor. An aluminum surface passivation treatment based on oxygen plasma is developed in order to circumvent the undesired effects of oxidation and corrosion usually found in aluminum-based biosensors. Immersion tests in deionized water and device simulations are used to evaluate the effectiveness of the passivation process. A label-free bioassay based on biotin analysis through biotin-functionalized dextran–lipase conjugates immobilized on the biosensor-passivated surface in aqueous media is performed as a proof of concept to demonstrate the suitability of these nanostructured aluminum films for biosensing.