Biosensing through White Light Reflectance Spectroscopy (WLRS) is based on monitoring the shift of interference spectrum due to the binding reactions occurring on top of a thin SiO2 layer deposited on a silicon chip. Multianalyte determinations were possible through scanning of a single sensor chip on which multiple bioreactive areas have been created. Nonetheless, the implementation of moving parts increased the instrumentation size and complexity and limited the potential for on-site determinations. Thus, in this work, a new approach, which is based on patterning the sensor surface to create areas with different SiO2 thickness, is developed and evaluated for multi-analyte determinations with the WLRS set-up. The areas of different thickness can be interrogated by a single reflection probe placed on a fixed position over the chip and the reflection spectrum recorded is deconvoluted to the spectra corresponding to each area allowing the simultaneous monitoring of the bioreactions taking place at each one of them. The combination of different areas thickness was optimized using chips with two areas for single analyte assays. The optimum chips were then used for the simultaneous determination of two mycotoxins, aflatoxin B1 and fumonisin B1. A competitive immunoassay format was followed employing immobilization of mycotoxin-protein conjugates onto the SiO2 of different thickness. It was found that the dual-analyte assays had identical analytical characteristics with the respective single-analyte ones. The detection limits achieved were 0.05 ng/mL for aflatoxin B1 and 1.0 ng/mL for fumonisin B1, with dynamic ranges extending up to 5.0 and 50 ng/mL, respectively. The sensor was also evaluated for the determination of the two mycotoxins in whole grain samples (wheat and maize). The extraction protocol was optimized and recoveries ranging from 85 to 115% have been determined. Due to lack of moving parts, the novel multi-analyte format is expected to considerably facilitate the built-up of a portable device for determination of analytes at the point-of-need.