Due to its biochemical and spectroscopic properties, phycocyanobilin (PCB) is of special interest for a variety of applications, but the production of PCB from Spirulina is complicated and costly. Here we report the assembly and regulation of the entire heme biosynthetic pathway for combinatorial biosynthesis of PCB in Escherichia coli. Our results shows that a plasmid containing bicistronic expression of hoxi and pcyA yields 6.64 mg/L PCB, which is higher than that of plasmid with one promoter for both genes (p < 0.01). Up-regulation of hemB, hemG, hemH, hemA(s), and hemL individually or simultaneously does not significantly improve the yield of PCB, but induces accumulation of 5-Aminolevulinic acid (ALA) and heme. Increasing the copy number of hoxi and pcyA genes slightly improves the production of PCB by enhancing the expression level of Hox1 and PcyA enzymes, suggesting that this two-step enzymatic reaction is the rate-limiting step for the production of PCB. This work provides new important insights into the regulatory mechanism of the heme biosynthesis pathway for production of valuable chemicals.