A novel method is demonstrated to detect Antifreeze proteins (AFPs) based on photoluminescence (PL) using porous silicon (PS) coated with silver as a substrate. Ag/PS substrate is obtained through immersion of PS in silver nitrate (AgNO3) solutions and is incubated with Rhodamine B (RB) as PL label. This substrate is easy to be fabricated and the pore size of PS is large enough for biological molecules to infiltrate, which is an ideal platform for biological molecule detection. Through functionalization used glutaraldehyde (GTA) and 4-(N-Maleimidomethyl) cyclohexane-1-carboxylicacid (Sulfo-SMCC) as cross-linkers separately, we test the role of the AFPs antibodies in selective capturing the AFPs antigen and explain the reason of the enhancement of PL intensity. The result shows a significant enhancement of the PL intensity of RB at around 590 nm due to the interaction of antibody-antigen competitive binding with AFPs. Therefore, the PL corresponding to RB was selected to detect the target AFPs and the PL intensity of RB proportional to the AFPs concentration. The detection limit was found to be 1.65 mu g/ml for AFPs when GTA was used as cross-linker, and the detection limit was 16.5 ng/ml with Sulfo-SMCC as cross-linker. (C) 2012 Elsevier B.V. All rights reserved.