Phytochromes, acting as red and far-red light receptors, orchestrate the developmental plasticity of plants to acclimate to the ambient environment throughout their entire life cycle. Recent structural biological studies have provided significant insights into the molecular bases of phytochrome-mediated light signaling in plants. Distinct approaches have been employed in these studies for phytochrome expression and purification. In our research, we utilized both bacteria and insect cells to separately synthesize the phytochromobilin (PΦB) molecule and express apophytochrome protein and subsequently purify the holophytochrome protein from the mixtures of the two expression systems. Here, we detail the procedures of this strategy, including plasmid construction, PΦB biosynthesis, apophytochrome expression, holophytochrome purification, and characterization of the phytochrome proteins. This approach can serve as an essential supplement to existing phytochrome purification methodologies, and we hope that investigators will benefit from it in achieving successful purification of the phytochromes they are interested in.