The single-layer porous silicon is prepared by electrochemistry etching method, which is used as an immunosensor for determining recombinant mouse zona pellucida 3 fusion protein (r-mZP3) by Raman spectroscopy analysis at room temperature. The molecule binding increases the effective optical thickness (EOT), and thus the Raman spectrum intensity decreases. The concentration and variation of Raman intensity show a good linear quantitative relation. The excellent sensing performance could open the way to a new family of optical sensors for biological standardization.