The phycobiliproteins (PBSs) are large pigment proteins found in certain algae that play a central role in harvesting light energy for photosynthesis. Phycocyanin (PC) is one type of PBSs that gains increasing attention owing to its various biological and pharmacological properties. In this paper, an expression vector containing five essential genes in charge of biosynthesis of cyanobacterial C-phycocyanin (C-PC) holo-alpha subunit (holo-CpcA) was successfully constructed resulting in over-expression of a fluorescent holo-CpcA in E. coli BL21. The vector harbored two cassettes: one cassette carried genes hox1 and pcyA required for conversion of heme to phycocyanobilin (PCB), and the other cassette carried cpcA encoding CpcA along with cpcE and cpcF both of which were necessary and sufficient for the correct addition of PCB to CpcA. The vector system contained a His-tag for protein purification. The purified protein showed correct molecular weight on SDS-PAGE gel and emitted orange fluorescence by UV excitation. The maximum peak of absorbance spectrum was at 623 nm, and the maximum peak of fluorescence emission and excitation were at 648 and 633 nm, respectively, which were similar to those of native C-PC. This study provides an efficient method for large-scale production of the fluorescent holo-CpcA in biotechnological applications.