A rapid and low cost photoluminescence (PL) immunosensor for the determination of low concentrations of Aflatoxin B1 (AFB1) has been developed. This immunosensor was based on porous silicon (PSi) covered by thin gold layer (Au) and modified by antibodies against AFB1 (anti-AFB1). PSi layer was formed on silicon substrate, then the surface of PSi was covered by 30 nm layer of gold (PSi/Au) using electrochemical and chemical deposition methods and in such ways PSi/Au_(El.) and PSi/Au_(Chem.) structures were formed, respectively. In order to find PSi/Au the most efficiently suitable for PL-based sensor design, structure several different PSi/Au_(El.) and PSi/Au_(Chem.) structures were designed while using different conditions for electrochemical or chemical deposition of gold layer. It was shown that during the formation of PSi/Au structure crystalline Au nanoparticles uniformly coated the surface of the PSi pores. PL spectroscopy of PSi/Au nanocomposites was performed at room temperature and it showed a wide emission band centered at 700 nm. Protein A was covalently immobilized on the surface of PSi/Au_(El.) and PSi/Au_(Chem.) forming PSi/Au_(El.)/Protein-A and PSi/Au_(Chem.)/Protein-A structures, respectively. In the next step PSi/Au_(El.)/Protein-A and PSi/Au_(Chem.)/Protein-A structures were modified by anti-AFB1 and in such way a structures (PSi/Au_(El.)/Protein-A/anti-AFB1 and PSi/Au_(Chem.)/Protein-A/anti-AFB1) sensitive towards AFB1 were designed. The PSi/Au_(El.)/Protein-A/anti-AFB1- and PSi/Au_(Chem.)/Protein-A/anti-AFB1-based immunosensors were tested in a wide range of AFB1 concentrations from 0.001 upon 100 ng/ml. Interaction of AFB1 with PSi/Au_(El.)/Protein-A/anti-AFB1- and PSi/Au_(Chem.)/Protein-A/anti-AFB1-based structures resulted PL quenching. The highest sensitivity towards AFB1 was determined for PSi/Au_(El.)/Protein-A/anti-AFB1-based immunosensor and it was in the range of 0.01–10 ng/ml. The applicability of PSi/Au-based structures as new substrates suitable for PL-based immunosensors is discussed.