Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB's antioxidation, anti-inflammatory and anti-cancer properties, it has been utilized in the food, pharmaceutical and cosmetic industries. Currently, the extraction of PCB from involves complex processes, which has led to increasing interest in the biosynthesis of PCB in . However, the PCB titer remains low because of the poor activity of key enzymes and the insufficient precursor supply. Here, the synthesis of PCB was firstly improved by screening the optimal heme oxygenase (HO) from BP-1(HO) and PCB: ferredoxin oxidoreductase from sp. PCC6803 (PcyA). In addition, based on a rational design and the infrared fluorescence method for high-throughput screening, the mutants of HO(F29W/K166D) and PcyA(D220G/H74M) with significantly higher activities were obtained. Furthermore, a DNA scaffold was applied in the assembly of HO and PcyA mutants to reduce the spatial barriers, and the heme supply was enhanced via the moderate overexpression of and , resulting in the highest PCB titer (184.20 mg/L) obtained in a 5 L fermenter. The strategies applied in this study lay the foundation for the industrial production of PCB and its heme derivatives.