The recombinant expression of phycocyanobilin (PCB) was carried out in Escherichia coli , and the best fermentation conditions of recombinant E . coli biosynthesized PCB are optimized in the response surface methodology to improve PCB production. The recombinant PCB is extracted, isolated, and purified by methanol and chloroform extraction. Recombinant PCB is validated in UV-vis spectroscopy, high-pressure liquid chromatography, and mass spectrometry. In addition, the anti-oxidant activities of the recombinant PCB are determined. The best induction conditions that optimized by Design Expert 8.0 software include: lactose concentration 4 mmol/L, induction temperature 24.69℃, induction time 4.6 h, and induction duration 13.57 h, under which the PCB expression level reached approximately 13 mg PCB/L, which is more than four times of previously reported 3 mg PCB/L. The maximum absorption peak of the recombinant PCB is located at 680 nm with a high fluorescence intensity of 470 nm. The recombinant PCB has a good ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals.