The presence of carcinogenic aflatoxins in food and feed products is a major worldwide problem. To date, the aflatoxin contamination can only be detected by the use of destructive sample-based chemical analyses. Therefore, we developed an optical setup able to detect the localized aflatoxin contamination in individual maize kernels, on the basis of one- and two- photon induced fluorescence spectroscopy. Our developed optical configuration comprises a tunable titanium-sapphire laser (710nm-830nm) in combination with second harmonic wavelength generation (355nm-415nm), enabling the measurement of both one- and two-photon induced fluorescence spectra. Moreover, an accurate scanning of the kernel’s surface was induced by the use of automated translation stages, allowing to study the localized maize contamination. First, the operation of the setup is validated by the characterization of pure aflatoxin B1 powder. Second, the fluorescence spectra of healthy (< 1ppb aflatoxin B1) and contaminated maize kernels (>70ppb aflatoxin B1) were measured, after excitation with 365nm, 730nm, 750nm and 780nm. For both the one- and two- photon induced fluorescence processes, the presence of the aflatoxin inside the contaminated maize kernels influenced the intrinsic fluorescence signals. Based on the fluorescence spectrum between 400nm and 550nm, we defined a detection criterion to identify the contaminated maize kernels. Furthermore, we demonstrate the sensing of the localized contamination level, indicating both contaminated maize kernels with a high contamination level in a limited surface area (as small as 1mm2) as with a lower contamination spread over a large surface area (up to 20mm2). As a result, our developed measurement methodology allows the identification of the localized aflatoxin contamination, paving the way to the non-destructive, real-time and high-sensitive industrial scanning-based detection of aflatoxins in food products.