Aflatoxins are extremely toxic metabolites from Aspergillus species that can adulterate a wide range of human foodstuff. Herein, we propose a novel assay designed as an analytical test for aflatoxin B1 and G1 (AFB1 and AFG1, respectively) that could represent an alternative screening technique for this class of mycotoxins. The approach for the determination of these toxins is based on surface plasmon resonance using neutrophil porcine elastase as a "bait" for these aflatoxins. The selection and optimization of the analytical procedure involved a preliminary investigation on the type of inhibition by AFB1: the level of the protease inhibition exerted by AFB1 depended upon the incubation time and the concentration of the binding partners, showing the competitiveness and the reversibility of the inhibition. A posteriori, the nature of the interaction granted a rapid analysis, a single detection test requiring only a few minutes. For the development of the assay, the experimental conditions were evaluated and optimized with both calibration solution and aflatoxin-spiked samples. To apply this method to aflatoxin-contaminated maize, a rapid solid-phase extraction treatment was developed. The proposed assay for AFB1 and AFG1 was validated by comparison with both a chromatographic reference method and a standard enzyme linked immunosorbent assay procedure. This enzyme-based biosensor represents a new approach for the detection of aflatoxins based on the reversible interaction between a blocked macromolecule and a soluble ligand, having the major advantages in the relative rapidity, the reusability of the capturing surface, and low cost per single test.