A barcode semiquantitative lateral flow immunochromatographic strip for prostate acid phosphatase (PAP) was developed, in which the monoclonal antibody specific for PAP was labeled to gold nanoparticle and another monoclonal antibody was immobilized on nitrocellulose membrane in the barcode fashion respectively. Based on the stepwise capture of analyte, the system expresses the concentration of PAP in nanogram range as four distinct ladder bars in 30 min, therefore, which could be detected directly by naked eye or image analyzer. Serum PAP from 65 patients was detected with this method and compared with enzyme linked immunosorbent assay (ELISA). There is a good agreement between the methods. Its easily readable result, and also its simplicity and low cost offers an alternative for testing PAP. By incorporating with different specific antibody, the assay can be further extended to detect a variety of analytes with clinical importance.