The development of fluorescent tools with desired fluorescence and efficient targeting is of great importance in the high-throughput immunoassay. Here we report combinational biosynthesis of new dual-functional streptavidin-phycobiliproteins (SA-PBPs) in Escherichia coli by fusing streptavidin with phycobiliproteins. These recombinant proteins can achieve a purity over 95% after one-step purification, and their maximum absorption and fluorescence emission wavelength are at 556 nm and 568 nm for SA-PCA-PEB (streptavidin-phycocyanin a subunit-phycoerythrin), and 624 nm and 646 nm for SA-PCA-PCB (streptavidin-phycocyanin a subunit-phycocyanobilin), respectively. The potential application of these dual-functional SA-PBPs in immunoassay was evaluated using "sandwich" ELISA method for detection of two biomarkers of liver cancers, i.e. alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). As a result, both SA-PCA-PCB and SA-PCA-PEB showed a good linear function with AFP & CEA within 0-50 ng/ml concentrations. Their limits,of detection (LODs) for AFP and CEA were 0.25 ng/mL and 0.28 ng/ml using SA-PCA-PEB, and 1.01 ng/ml and 1.12 ng/ml using SA-PCA-PCB respectively. These results indicate that these novel dual-functional SA-PBPs are useful tools for a wide variety of immunoassay, and may have the advantages in their higher expandability and compatibility with existing and future immunoassay technologies.