In the current research, a porous silicon/zinc oxide (PSi/ZnO) nanocomposite produced by a combination of metal-assisted chemical etching (MACE) and atomic layer deposition (ALD) methods is presented. The applicability of the composite for biophotonics (optical biosensing) was investigated. To characterize the structural and optical properties of the produced PSi/ZnO nanocomposites, several studies were performed: scanning and transmission electron microscopy (SEM/TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), diffuse reflectance, and photoluminescence (PL). It was found that the ALD ZnO layer fully covers the PSi, and it possesses a polycrystalline wurtzite structure. The effect of the number of ALD cycles and the type of Si doping on the optical properties of nanocomposites was determined. PL measurements showed a “shoulder-shape” emission in the visible range. The mechanisms of the observed PL were discussed. It was demonstrated that the improved PL performance of the PSi/ZnO nanocomposites could be used for implementation in optical biosensor applications. Furthermore, the produced PSi/ZnO nanocomposite was tested for optical/PL biosensing towards mycotoxins (Aflatoxin B1) detection, confirming the applicability of the nanocomposites.