Phycocyanobilin (PCB) is a blue pigment with antioxidant, anti-inflammatory, and anticancer properties. It is used in the medical and cosmetic industries. In this study, a high-expression plasmid, pET-30a-PCB, was constructed for expression of PCB in Escherichia coli BL21(DE3). The PCB was analyzed using UV-visible absorption spectrum, MALDI-TOF-MS, and fluorescence spectra. The stability and half-life of PCB in different serum were determined. The yield of PCB was optimized through single-factor and orthogonal experiments. The optimal expression conditions were determined as a lactose concentration of 5 mmol/L, an induction time of 8 h, an induction temperature of 27 °C, and an induction duration of 22 h. PCB yield of 6.5 mg/L was achieved and subsequently purified using nickel-affinity chromatography. The purified PCB was quantified indirectly using Hist-tag ELISA detection, and the concentration was 11.66 μg/L. In the range of 0-33 μg/mL, the total antioxidant capacity and reducing the capacity of PCB were stronger than Vitamin E (Ve), with 1,1-diphenyl-2-picrylhydrazil (DPPH) scavenging reaching up to 87.07%, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS) scavenging up to 100%, hydroxyl radicals (·OH) scavenging up to 64.19%, hydrogen peroxide (HO) scavenging up to 78.75%, This study provides theoretical evidence for PCB as a potent antioxidant.